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The Pulmonary Environment Promotes Th2 Cell Responses After Nasal-Pulmonary Immunization with Antigen Alone, but Th1 Responses Are Induced During Instances of Intense Immune Stimulation
Harlan P. Jones, Lisa M. Hodge, Kohtaro Fujihashi, Hiroshi Kiyono, Jerry R. McGhee and Jerry W. Simecka
J Immunol October 15, 2001, 167 (8) 4518-4526; DOI: https://doi.org/10.4049/jimmunol.167.8.4518
Harlan P. Jones
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107;
Lisa M. Hodge
Kohtaro Fujihashi
Departments of Microbiology and Oral Biology, Immunobiology Vaccine Center, University of Alabama, Birmingham, AL 35294; and
Hiroshi Kiyono
Departments of Microbiology and Oral Biology, Immunobiology Vaccine Center, University of Alabama, Birmingham, AL 35294; and Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
Jerry R. McGhee
Jerry W. Simecka
The purpose of this study was to determine the nature of the CD4+ Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4+, and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-γ, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-γ) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4+ Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1α (MIP-1α) and MIP-1β mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1β expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.
Induction of both mucosal and systemic (circulating) immunity is optimal for protection and recovery from respiratory infections, and may be critical determinants in pulmonary disease. Ab responses in the circulation protect against pulmonary infections and disease (1, 2, 3). Presumably, IgG Abs cross the alveolar-capillary barrier into the alveoli, where they provide resistance to infection. Importantly, the airway epithelium is the initial site for many infections (e.g., Mycoplasma and influenza virus) in the upper and lower respiratory tracts. Mucosal IgA responses are involved in protection of these surfaces from infection and perhaps in recovery from disease (2, 4, 5, 6, 7, 8, 9). For example, we previously showed that immune responses can contribute to the severity of pulmonary inflammatory responses in Mycoplasma respiratory disease, while at the same time preventing dissemination of the infection to extrapulmonary sites (10). Furthermore, immunity is central to the development of respiratory allergies and asthma (11, 12), in which infectious agents and environmental factors appear to affect the susceptibility or severity of these conditions (13, 14, 15, 16, 17, 18, 19, 20, 21). Thus, immunity can have both beneficial and detrimental effects on respiratory disease, and it is critical to understand the factors that influence immune responses that can impact the progression and prevention of respiratory diseases.
Nasal immunization is commonly used to induce immunity along the respiratory tract. This route of immunization results in mucosal IgA and systemic IgG Ab responses, which has the advantage of protecting the entire respiratory tract from respiratory infections (22, 23, 24, 25, 26, 27). Importantly, nasal immunization also has the potential to elicit immunopathologic responses (28, 29). Nasal immunization can result in the generation of Ag-specific IgE Ab responses. Mucosal surfaces are associated with generation of IgE responses, which play essential roles in respiratory allergies and asthma (30). IgE-mediated responses may also contribute to the pathogenesis of infectious diseases, including Mycoplasma and viral pneumonias (13, 31, 32), and interestingly, infectious agents may actually increase the risk or severity of asthma (14, 16). Furthermore, the use of the mucosal adjuvant cholera toxin (CT)3 resulted in significant enhancement of IgE Ab responses, with concomitant mucosal IgA and serum IgG Ab responses (28, 29). When CT is used as an adjuvant, there is a massive infiltration of mononuclear cells into the lungs. Thus, an understanding of the mechanisms for these responses will potentially provide insights into the induction of immunity during respiratory infections and diseases, as well as suggesting new approaches for mucosal vaccines development.
Due to their central role in immunity, alterations in the intensity or the type of CD4+ Th cell responses can have a profound effect on respiratory disease. Th cells may be divided into two major functional populations, in which Th1 cells promote cell-mediated and inflammatory responses through their production of cytokines, such as IL-2 and IFN-γ (33). For example, Th1 responses are critical in controlling mycobacterial pulmonary infections (34, 35, 36). In contrast to Th1 cells, Th2 cells regulate Ab responses through secretion of IL-4, IL-5, IL-10, and IL-13 (33). In the gastrointestinal tract, Th2 cells are the dominant resident Th cell population (37, 38), and presumably, Th2 cells are critical along mucosal surfaces of the respiratory tract as well. The Th2 cytokines are intimately involved in the generation of mucosal IgA Ab responses (39). Importantly, IL-4 produced by Th2 cells also promotes the generation of IgE Ab responses (33, 40), and therefore, in addition to mucosal IgA responses, Th2 cells are major mediators of allergies and asthma (41, 42, 43, 44, 45). These responses appear to also contribute to the pathology of some respiratory infections (46, 47, 48). Past studies suggest that Th2 cell responses are often polarized due to cytokines promoting one response and inhibiting the other (49, 50, 51). However, recent studies suggest that Th1 cells may also contribute to the pathogenesis of the inflammatory responses associated with the Th2 cell-mediated disease, asthma (45). Thus, both Th cell subsets can be involved in the pathogenesis of inflammatory diseases of the respiratory tract. The purpose of the present studies was to assess Th cell subset responses in the lung after nasal immunization. Our results provide important insights into the mechanisms of pulmonary immunity in health and disease, and perhaps reveal new approaches for the development of mucosal vaccines and immunotherapy.
Viral-free female BALB/c mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN). All mice were used between 8 and 12 wk of age. All mice were maintained in sterile microisolator cages, and given sterile water and food ad libitum. Before experimental manipulation, mice were anesthetized with an i.m. injection of ketamine-xylazine. For nasal-pulmonary immunization, mice were allowed to inhale 25 μl inoculum that was placed upon the nares (29, 52). This volume of inoculum results in the deposition of Ag in both nasal passages and lung (28).
Immunogens
Philippines influenza split virus vaccine (H3N2) was provided by M. W. Harmon, Connaught Laboratories (Swiftwater, PA). CT was purchased from List Biological Laboratories (Campbell, CA).
Mononuclear cells were isolated from lungs, as previously described (32, 53, 54). Lungs were perfused with PBS without magnesium or calcium to minimize contamination of the final lung cell population with those from blood. The lungs were separated into individual lobes and finely minced. The tissues were suspended in RPMI 1640 (HyClone Laboratories, Logan, UT) medium containing 300 U/ml Clostridium histolyticum type I collagenase (Worthington Biochemical, Freehold, NJ), 50 U/ml DNase (Sigma, St. Louis, MO), 10% FBS (HyClone Laboratories), HEPES, and antibiotic/antimycotic solution (Life Technologies, Grand Island, NY). The tissues were incubated at 37°C while mixing on a Nutator (Fisher Scientific, Pittsburgh, PA) for 90–120 min. During the incubation period, the tissue was vigorously pipetted every 20 min. After incubation, the digestion mixture was passed through a 250-μm nylon mesh to remove undigested tissue. Mononuclear cells were purified from cell suspensions by density gradient centrifugation using Lympholyte M (Accurate Chemicals, Westbury, NY).
Spleen cells and lower respiratory (mediastinal and hilar) lymph nodes were isolated after centrifugation of cell suspensions, followed by red cell removal using ammonium chloride potassium lysis buffer, as previously described (55).
Immunofluorescent characterization of lymphocyte populations
Three-color immunofluorescent staining was performed to identify T cell populations using PE-labeled anti-murine CD3 mAb (KT3; Beckman Coulter, Fullerton, CA), PerCP-labeled anti-CD4 mAb (0.5 μg/ml, L3T4, RM4-5; BD PharMingen, San Diego, CA), and FITC-labeled anti-CD8 mAb (0.2 μg/ml, Lyt-2, 53-6.7; BD PharMingen). Briefly, 2 × 106 cells/tube were incubated with purified 2.4G2 mAb (BD PharMingen) for 5 min at 4°C to reduce nonspecific binding of FcII/III receptors before fluorescent Ab staining. The cells were incubated for 30 min at 4°C with 250 μl fluorescent Ab. The cells were washed in staining buffer (Mg2+-free, Ca2+-free PBS with 0.05% sodium azide, 1% FBS (HyClone Laboratories)) and fixed with 4% paraformaldehyde solution for 30 min, and the cells were then resuspended in staining buffer until analyzed.
The cells were analyzed using an EPICS XL-MCL flow cytometer (Beckman Coulter). Data collection was done using System 2 software (Beckman Coulter), with further analysis done using Expo 2 analysis software (Beckman Coulter). Lymphocyte gates and detector voltages were set using unstained (control) lung and splenic cells, and stained cell populations were seen as distinct peaks or clusters of cells. The proportion of each cell population was expressed as the percentage of the number of stained cells. To determine the total number of a specific lymphocyte population, their percentage was multiplied by the total number of lymphocytes isolated from a particular tissue.
In vitro depletion and isolation of T lymphocyte populations
A 100-μl aliquot of mouse anti-CD4 and/or anti-CD8 mAb-labeled magnetic beads (Dynabeads mouse CD4, mouse CD8; Dynal, Lake Success, NY) was added directly to lung and splenic cell suspensions at a final concentration of 1 × 107 cells/4 × 107 beads. Lymphoid cells were incubated at 4°C for 20 min on a rotator. After incubation, tubes containing lymphocytes were placed on a magnet (MPC magnet; Dynal) for 2 min, and both positive and negative fractions were collected for further studies. Confirmation of cell purity was determined using flow cytometry. Cell fractions with a depletion of >98% were used for subsequent experimentation.
Polyclonal stimulation of CD3+ lymphocytes
A 50-μl aliquot of a 10 μg/ml concentration of purified anti-CD3 mAb (145-2C11; BD PharMingen) diluted in PBS was added to a sterile 96-well flat-bottom microtiter plate and incubated overnight at 4°C. In some experiments, anti-CD28 mAb (37.51; BD PharMingen) was added to the cell cultures at a concentration of 1.25 μg/ml. Plates were gently washed with sterile RPMI 1640 culture medium (HyClone Laboratories). Whole and purified lymphocyte fractions of lung and splenic mononuclear cells were placed in wells suspended in 100 μl culture media at a final concentration of 2 × 106 cells/ml and incubated for 4 days at 37°C and 5% CO2 in air. Supernatants were collected and stored at −80°C until assayed for cytokine levels.
Cytokine assays
The levels of cytokine were measured by capture ELISA. Murine IL-4 levels were measured using OptEIA IL-4 ELISA set (BD PharMingen), while murine IFN-γ levels were determined using mouse IFN-γ MiniKit (Endogen, Woburn, MA). Easy-wash 96-well flat-bottom microtiter plates (BD Biosciences, Bedford, MA) were coated overnight at 4°C with 100 μl mAb specific for either murine IL-4 or IFN-γ diluted in 0.1 M Na2HPO4, pH 9. Plates were washed and blocked with 200 μl PBS/Tween 20 supplemented with 10% FBS for 30 min. Following a PBS/Tween 20 wash, 100 μl sample supernatants were placed into the appropriate wells and incubated overnight at 4°C. Plates were washed three times with PBS/0.05% Tween, and 100 μl biotinylated rat anti-mouse cytokine (IL-4 or IFN-γ) mAb was added to each well and incubated overnight at 4°C. To reveal the reaction, avidin-HRP and 3,3′5,5′-tetramethylbenzidine substrate (Moss, Pasadena, MD) were used. Plates were read using an MX80 plate reader (Dynatech, Chantilly, VA) at an absorbance of 630 nm. One hundred microliters of 0.25 M HCl were also added to the reaction as needed to increase the sensitivity of reaction, and read at an absorbance of 450 nm. Cytokine levels were determined by comparison with standard curves generated from murine recombinant cytokines (IL-4 and IFN-γ; BD PharMingen) after log/log quadratic linear regression analysis using Revelation 2.0 software (Dynatech).
RNA isolation from lungs
Total RNA was isolated from the entire lungs of mice using the Ultraspec-II RNA Isolation System (Biotecx Laboratories, Houston, TX), which is based on a previously described method (56). Briefly, the lungs were homogenized in the Ultraspec-II RNA reagent using a PRO 200 homogenizer (PRO Scientific, Monroe, CT). Chloroform was added to the homogenate and centrifuged at 12,000 × g (4°C) for 30 min. The RNA was precipitated by adding isopropanol to the aqueous phase and centrifuging the samples at 12,000 × g (4°C) for 10 min. The RNA pellet for each sample was washed twice with 75% ethanol by vortexing and subsequent centrifugation for 5 min at 7,500 × g , and then resuspended in diethylpyrocarbonate-treated water. The concentration and quality of RNA in each of the samples were determined spectrophotometrically (GeneQuant II; Pharmacia Biotech, Piscataway, NJ) and by gel electrophoresis. The RNA samples were stored at −80°C until used.
Cytokine mRNA detection by RT-PCR
RT-PCR was performed using 100 ng RNA for each sample, as previously described (57). The sequences of the primers and the size of the resulting PCR fragments (in parentheses) for IL-2, IL-4, IL-5, IFN-γ, and the housekeeping gene, β2-microglobulin (β2mGL), are as follows (58): IL-2 (227 bp), 5′-GACACTTGTGCTCCTTGTCA and 5′-TCAATTCTGTGGCCTGCTTG; IL-4 (216 bp), 5′-TCGGCATTTTGAACGAGGTC and 5′-GAAAAGCCCGAAAGAGTCTC; IL-5 (201 bp), 5′-TCACCGAGCTCTGTTGACAA and 5′-CCACACTTCTCTTTTGGCG; IFN-γ (227 bp), 5′-GCTCGAGACAATGAACGCT and 5′-AAAGAGATAATCTGGCTCTGC; and β2mGL (222 bp), 5′-TGACCGGCTTGTATGCTATC and 5′-CAGTGTGAGCCAGGATATAG. β-Chemokine primers used were as described in a previous study (59).
The increase in expression of cytokine mRNA after immunization was determined by the number of cycles of amplification that resulted in little or no PCR product for each cytokine in total lung RNA from sham-inoculated, control mice, as previously described (57, 59). For IFN-γ, IL-5, and β2mGL, the samples were amplified for 30 cycles, and for IL-2 and IL-4, the samples were run for 35 cycles. The PCR products were separated on 1.8% agarose gels and stained with ethidium bromide. Gels were visualized using the α Image 2000 Documentation and Analysis System (Alpha Innotech, San Leandro, CA). The intensity of each band was determined using densitometry, and the relative cytokine mRNA reactions were compared after normalization to the housekeeping gene, β2mGL.
Cytokine RNase protection assay
RiboQuant, Multiprobe Ribonuclease Protection Assay System (BD PharMingen), was used for T cell cytokine mRNA detection and quantitation. RNA probes specific for T cell-specific cytokine mRNAs (mCK-1 multiprobe template set; BD PharMingen) were synthesized using T7 RNA polymerase, GACU nucleotide pool, and [α-23P]UTP (37°C for 1 h). The RNA probes were subsequently purified and quantitated for radiolabel incorporation. A 20-μg aliquot of total lung RNA was hybridized in solution to the radiolabeled probes for 16 h at 56°C. After hybridization, the samples were treated with RNase A to degrade unhybridized RNA probes, and the RNase A activity was subsequently inactivated using proteinase K, followed by phenol/choroform:isoamyl extraction to purify nucleic acids. The hybridized probes were resolved on a 5% polyacrylamide sequencing gel. The radiolabeled probes were visualized by exposure to x-ray film. Exposure times were varied to ensure visualization of probes and linearity of detection. The identity of the protected probes was verified using undigested probes, as m.w. markers, and comparing their sizes to expected m.w. For densitometry, x-ray films were analyzed using the α Image 2000 Documentation and Analysis System (Alpha Innotech). Probe intensities were compared between samples after normalization using the intensity of band produced by the housekeeping gene, L32. Yeast tRNA was used as a negative control to verify hybridization and digestion of radiolabeled antisense probes.
Ag-specific in vitro stimulation of mononuclear cells
Lymphoid cells were cultured in 96-well round-bottom microtiter plates in RPMI 1640 (HyClone Laboratories) supplemented with 5% FBS (HyClone Laboratories), HEPES, 10 U/ml rIL-2 (BD PharMingen), antibiotic/antimycotic solution (Life Technologies), and 50 μM 2-ME (Life Technologies). Lymphoid cells were stimulated at 37°C and 5% CO2. Cells were stimulated with or without 2 μg/ml dialyzed influenza Ag in a final volume of 200 μl/well of culture media at a cell concentration of 2 × 106 cells/ml. Supernatants were collected 4 days later and stored at −80°C until assayed for cytokine levels.
Data were evaluated by ANOVA, followed by Fisher protected least significant difference multigroup comparison, Tukey's multigroup comparison, or Mann-Whitney U tests using Bonferroni-adjusted probabilities. These analyses were performed using the SYSTAT (SYSTAT, Evanston, IL) or StatView (SAS Institute, Cary, NC) computer programs. When appropriate, data were logarithmically transformed before statistical analysis, and confirmed by a demonstrated increase in power of the test after transformation of the data. A p value of less than or equal to 0.05 (p ≤ 0.05) was considered statistically significant. If data were analyzed after logarithmic transformation, the antilog of the means and SEs of the transformed data was used to present the data, and are referred to as the geometric means (x/÷ SE).
Resident pulmonary CD4+ Th cells are predominantly of a Th2 type
To determine the resident lymphocyte environment in the lungs and spleens of naive mice, mononuclear cells were isolated from naive mice. Cells from lung and spleen were stained for CD3, CD4, and CD8 surface expression. The percentages of total CD3+, CD3+CD4+, and CD3+CD8+ T cells were determined using flow cytometry. The major T cell subset in the lungs was CD3+CD4+ (73.4 ± 3.8% of total T cells), while CD3+CD8+ T cells represent approximately 20% of the entire pulmonary T cell population. A similar T cell distribution was seen in spleen.
To examine cytokine production by resident naive lung lymphocytes, we measured Th1 (IFN-γ) and Th2 (IL-4) cytokine production by isolated T cells after in vitro polyclonal activation. Lung cells and splenocytes were collected from naive mice and cultured in wells coated with anti-CD3 mAb. After 4 days in culture, cytokine levels in culture supernatants were measured. Lung cells produced significantly higher amounts of IL-4 than did splenocytes (Fig. 1⇓). In contrast, IFN-γ production was significantly higher in splenic cell cultures than in cells isolated from the lungs (Fig. 1⇓).
IL-4 and IFN-γ cytokine production by naive resident lung and splenic lymphocytes. Lung and spleen mononuclear cells, isolated from naive mice, were cultured in wells coated with (Stimulated) or without (Unstimulated) anti-CD3 mAb. After 4 days in culture, cytokine levels (picograms per milliliter) in culture supernatants were measured using ELISA. Vertical bars and error bars represent mean ± SE (n = 3). ∗, Significantly (p ≤ 0.05) higher level of cytokines detected within culture supernatants.
To determine the contribution of CD4+ and CD8+ T cells to in vitro cytokine production, lung CD4+ and/or CD8+ T cell populations were depleted (>98% depletion) from naive whole lymphocyte populations. After depletion, lymphocytes were stimulated with anti-CD3 mAb, and the levels of cytokines in culture supernatants were determined (Fig. 2⇓). Depletion of CD4+ T cells markedly reduced IL-4 production by lung cell suspensions. On the other hand, depletion of CD8+ T cells had no effect on IL-4 production by lung lymphocytes. In contrast, CD4+ T cell depletion had no significant effect on IFN-γ levels when compared with unfractionated lung cells. Simultaneous depletion of both CD4+ and CD8+ T cell populations resulted in small amounts of IL-4 or IFN-γ cytokine production by lung cells. Similar results were obtained when anti-CD28 mAb was used with anti-CD3 mAb to stimulate naive T cells (data not shown).
The contribution of lung CD4+ and CD8+ T cells to in vitro cytokine production. CD4+ and/or CD8+ lung lymphocyte populations were depleted (>98% depletion, as confirmed using flow cytometry) from naive whole lymphocyte populations. Lung cells were stimulated with (Stimulated, filled bar) or without (Unstimulated, hatched bar) anti-CD3 mAb, and the levels of IL-4 and IFN-γ cytokines in culture supernatants were determined using ELISA. Results are represented as the percentage of cytokine production detected within the culture supernatants of whole (undepleted (None)) lung cells. Vertical bars and error bars represent the mean ± SE (n = 3). ∗, Significant (p ≤ 0.05) reduction in detectable levels of IL-4 and IFN-γ cytokine production from undepleted (None) lung cells.
Cytokine responses in lungs after nasal instillation of influenza Ag alone or in combination with CT
We next assessed CD4+ Th cell cytokine mRNA expression in lungs after nasal-pulmonary immunization with influenza Ag and CT as adjuvant. Mice were given either Ag alone (7.5 μg) or with CT (0.1 μg) nasally on days 0 and 7. Three days after the second immunization, lung RNA was isolated, and Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokine mRNA expression was determined by RT-PCR. After immunization with influenza Ag only, a significant induction of IL-4 and IL-5 mRNA expression was noted when compared with sham-immunized mice, but no significant increases in IL-2 or IFN-γ mRNA were seen (Fig. 3⇓). In addition, using the adjuvant CT resulted in induction of both Th2 (IL-4 and IL-5) and Th1 (IL-2 and IFN-γ) cytokine mRNA expression (p ≤ 0.05).
Th1 and Th2 cytokine mRNA expression after nasal immunization with influenza vaccine (FLU) alone or in combination with cholera toxin (FLU + CT). Total RNA was isolated from the lungs of mice 3 days after secondary nasal immunization (day 7) either with PBS (hatched bar), influenza vaccine alone (7.5 μg) (filled bar), or in combination with CT (0.1 μg) (open bar). Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokine mRNA expression was examined in the lungs using RT-PCR. Relative differences in mRNA expression of cytokines between groups of mice were determined by the relative increase in the ratio of cytokine to the housekeeping gene β2mGL. ∗, Significant (p ≤ 0.05) difference in band intensity from PBS-treated mice; ∗∗, significant difference (p ≤ 0.05) in band intensity from all other groups of mice. Results are based on the geometric mean (x/÷ SE, n = 6).
Using a quantitative RNase protection assay, the levels of Th cytokine mRNAs were further compared between mice immunized with Ag alone and those mice given Ag plus CT as adjuvant. The mRNAs encoding the Th2 cytokines, IL-4, IL-5, IL-6, IL-10, and IL-13, were significantly higher in lungs of mice given influenza vaccine with CT than in those given influenza Ag alone (Fig. 4⇓). The levels of IL-4 mRNA were 25-fold higher in mice immunized with influenza Ag and CT than in those given Ag alone (Fig. 4⇓a). The levels of IL-5, IL-6, IL-10, and IL-13 mRNA were 3- to 5-fold higher when CT was used as adjuvant (Fig. 4⇓b). Similarly, IFN-γ and IL-2 mRNA levels were significantly higher with the inclusion of CT as adjuvant. The levels of IFN-γ mRNA were 3-fold, while IL-2 mRNA was 2-fold higher when CT was used when compared with mice immunized with influenza Ag only. Thus, although Th2-type cytokines were induced in lungs after immunization with Ag alone, the inclusion of CT also enhanced the expression of all the Th cytokine mRNAs tested.
Th1 and Th2 cytokine mRNA expression after nasal immunization. Total RNA was isolated from the lungs of mice 3 days after secondary immunization either with PBS, influenza vaccine Ag (FLU), or vaccine Ag plus CT (FLU + CT). Relative differences in Th2 (A) and Th1 (B) mRNA expression of cytokines between groups of mice were determined by normalizing the cytokine band intensity with that of the housekeeping gene L32 for each sample. ∗, Significant (p ≤ 0.05) difference in band intensity from PBS-treated mice. Results are based on the geometric mean (x/÷ SE, n = 6).
Influenza-specific Th1 and Th2 cell responses in lungs of mice given CT nasally as adjuvant
To determine whether Ag-specific Th cell responses were generated, mice were given either Ag alone (7.5 μg) or with CT (0.1 μg) nasally on days 0 and 7. These dosages are consistent with our previous study (28). Three days after the second immunization, lung and spleen cells were isolated from these mice and stimulated with influenza Ag in vitro. Four days later, IL-4 and IFN-γ cytokines were measured in culture supernatants. IL-4 was detected in the supernatants from Ag-stimulated cultures isolated from mice immunized with Ag alone, whereas IFN-γ was not detected (Fig. 5⇓). In contrast, lung and spleen cells from mice immunized with Ag plus CT produced not only IL-4, but also IFN-γ.
Ag-specific IL-4 and IFN-γ cytokine production in mice using CT. Mononuclear cells were isolated from the lungs and spleens of control and immunized mice. Lung and splenic lymphocytes were stimulated in vitro with or without influenza Ag. Four days later, culture supernatants were collected, and IL-4 and IFN-γ levels (picograms per milliliter) were measured using ELISA. Vertical bars represent the mean ± the SE (n = 3). ∗, Significant (p ≤ 0.05) difference in cytokine levels from cells obtained from PBS-inoculated mice.
To analyze the contribution of CD4+ T cells in T cell cytokine responses, CD4+ and/or CD8+ T cells were depleted from lung lymphocytes before in vitro stimulation with Ag. Depletion of CD4+ T cells from lung lymphocytes collected from mice immunized with influenza Ag alone significantly reduced the levels of IL-4 from that of whole (undepleted) pulmonary cell isolates (Fig. 6⇓). Depletion of CD4+ T cells from lung cell isolates taken from mice immunized with CT resulted in a significant decrease in both IL-4 and IFN-γ. Depletion of lung CD8+ T cells also significantly reduced (p ≤ 0.05) IL-4 and IFN-γ levels when compared with Ag-stimulated whole cell cultures. The production of IL-4 and IFN-γ was diminished to almost undetectable levels in cultures depleted of both CD4+ and CD8+ T cells from immunized mice.
Ag-specific IL-4 and IFN-γ cytokine production after depletion of CD4+ and/or CD8+ lung cells collected from immunized mice. Lung cells from previously immunized mice were depleted of CD4+ and/or CD8+ T cells and placed in culture in the presence of influenza Ag. Four days later, levels of IL-4 and IFN-γ cytokines were evaluated using ELISA. Results are represented as the percentage of cytokine detected as compared with whole (undepleted (None)) cultures. Vertical bars and error bars represent the mean ± SE (n = 2). ∗, Significant (p ≤ 0.05) reduction in detected cytokine levels as compared with whole (None) cell cultures.
Nasal delivery of CT results in pulmonary CD4+ T cell expansion
Our previous studies (28, 29) showed a marked increase in mononuclear cells in the lungs of mice given CT as a mucosal adjuvant. To determine whether Th cells are a major component of the mononuclear cell infiltrate, mice were given a primary and booster immunization with influenza Ag alone or in combination with CT. Three days later, lung cells were isolated and cell numbers were determined. The percentage and numbers of lung CD4+ and CD8+ T cell populations were determined by flow cytometry.
The greatest effect on CD4+ T cells in lungs was seen when CT was included during nasal immunization. The numbers and percentages of CD3+CD4+ and CD3+CD8+ lung T cells from mice given Ag alone did not differ significantly from control mice. In contrast, there was a significant increase in the percentage of CD3+CD4+, but not CD3+CD8+, T cells isolated from lungs of mice immunized with Ag plus CT (Table I⇓). Furthermore, total numbers of CD3+CD4+ T cells were significantly higher in the lungs after nasal immunization with the mucosal adjuvant CT, whereas the numbers of CD3+CD8+ T cells did not increase (Fig. 7⇓). Thus, there was no major change in Th cells in lungs of mice after immunization with Ag alone; however, the inclusion of CT resulted in an overall increase in pulmonary Th cell numbers.
Numbers of CD4+ T cells increase in the lungs after nasal immunization with CT. Mice were given a primary and secondary immunization with Ag (FLU) alone or with CT (FLU + CT). Three days later, lung lymphocytes were isolated, and cell numbers were determined. The numbers of total T cells (CD3+) and CD3+CD4+ and CD3+CD8+ T cell populations were determined using flow cytometry. Data represent the percentage of increase in the total number of CD3+CD4+ and CD3+CD8+ cells based upon the percentage of CD3+ cells compared with PBS (unimmunized) mice. Vertical bars represent mean ± SE (n = 3). ∗, Significant (p ≤ 0.05) difference between immunized and unimmunized (PBS-inoculated) mice.
Table I.
T lymphocyte populations in pulmonary lymphocytes isolated from mice after nasal immunizationa
Nasal-pulmonary immunization induces expression of β-chemokine mRNA
We determined whether the increase in pulmonary cell numbers corresponded with increased β-chemokine expression, since these chemokines are chemotactic for T cells (60, 61, 62). Importantly, Th cell subsets differ in their responses to the β-chemokines, macrophage-inflammatory protein-1α (MIP-1α) and MIP-1β (63, 64, 65). Total RNA was isolated from lungs of mice 3 days after the second nasal-pulmonary immunization with influenza vaccine Ag alone or in combination with the mucosal adjuvant CT. The expression of MIP-1α, MIP-1β, monocyte chemoattractant protein-1, and RANTES mRNAs was analyzed by RT-PCR, as previously described (59). Significant induction of MIP-1β mRNA expression occurred in the lungs of immunized mice given Ag only or Ag plus CT; however, MIP-1β mRNA levels were significantly higher in mice immunized with Ag plus CT (Fig. 8⇓). In contrast, increases in MIP-1α mRNA expression were significant only in mice receiving Ag plus CT. No differences in monocyte chemoattractant protein-1 and RANTES mRNA expression were found in the lungs of naive mice and any group of immunized mice.
β-Chemokine mRNA expression after nasal immunization with CT. Three days after secondary immunization with PBS, influenza Ag alone, or influenza Ag in combination with CT, total RNA was isolated from mouse lungs. β-Chemokine mRNA expression was determined using RT-PCR. Relative differences in mRNA expression of cytokines between groups of mice were determined by comparing the ratio of cytokine mRNA with the housekeeping gene, β2mGL, mRNA. Vertical bars represent mean ± SE (n = 6). ∗, Significant difference from PBS-treated mice; ∗∗, significantly (p ≤ 0.05) higher band intensity than from all other groups of mice.
The purpose of the present study was to determine the Th cell subset responses generated in the lung after nasal-pulmonary immunization. In previous studies, we showed that nasal-pulmonary immunization resulted in the induction of Ag-specific IgE responses, and that use of CT markedly enhanced IgE Ab responses (28, 29). One also sees a massive infiltration of mononuclear cells into the lungs of BALB/c mice when CT is coadministered with Ag, but not when Ag is given alone. In preliminary studies, we also found a 2- to 3-fold increase in eosinophils in lung after nasal immunization with Ag plus CT. Based upon these past studies, we postulated that Th2 cell activity predominated after nasal-pulmonary immunization and contributed to the pulmonary inflammation associated with the use of CT (29). This was supported by previous studies demonstrating preferential Th2 cell responses to parasites in the BALB/c mouse model (66, 67). Furthermore, a recent study (68) suggested that nasal infection with a parasite in a Th1-dominant mouse strain (C57BL/6N) resulted in pulmonary Th2 cell activation. However, studies have also shown that Th1-type responses can contribute to airway inflammation in a murine model of asthma with BALB/c mice (45). Thus, the goal of these studies was to determine the degree of pulmonary Th1 cell activation that occurs in a Th2-biased environment.
First, we noted that CD4+ T cells were the major T cell population in the lungs of naive mice. After polyclonal stimulation of T cells using anti-CD3 Abs, the production of characteristic Th2 cytokine IL-4 was induced in culture. CD4+ Th cells were the major source of IL-4 as their depletion eliminated production of this cytokine. CD8+ T cells, not CD4+ T cells, were the source of IFN-γ produced in response to polyclonal stimulation. In contrast to lung lymphoid cells, preliminary studies suggested that splenic Th cells produced both Th1 (IFN-γ) and Th2 (IL-4) cytokines after anti-CD3 mAb activation (data not shown) or after Con A stimulation (69), indicating a fundamental difference in CD4+ Th cell populations residing in lungs and spleens. Thus, the resident CD4+ T cell population in lungs is of Th2 type, and was consistent with the idea that humoral immunity, and in particular mucosal Abs, played a major role in protection from respiratory pathogens.
Influenza-specific Th2 cell responses in lungs were indeed generated after nasal-pulmonary immunization with influenza Ag only. The Th2 cytokine mRNAs, IL-4 and IL-5, were readily detected in the lungs of mice after nasal immunization with Ag alone. In contrast, increases in neither IFN-γ nor IL-2 cytokine mRNA expression were noted. In addition, IL-4 was found in culture supernatants of lung cells from immunized mice stimulated in vitro with Ag, and CD4+ Th cells were the major source of IL-4 as their depletion eliminated IL-4 production. A similar IL-4 response was found in splenic lymphocytes, although the response from pulmonary lymphocytes was greater. It is now well established that IL-4 production is involved in mucosal immunity (39); in particular, IL-4 is an important factor in IgE production (70). In fact, our previous studies demonstrated that IgE responses are associated with nasal immunization (28, 29). Thus, influenza Ag introduced into the lung clearly generates almost solely Th2-type responses, reflecting the predominant resident Th2 environment.
The use of CT resulted in a significant enhancement of pulmonary CD4+ Th2 cell responses; however, there was also a Th1 component to the response. Our previous studies demonstrated that inclusion of CT greatly enhanced both systemic and respiratory Ab responses to Ag (28), and the present study shows that CT also enhances T cell responses in the lungs and spleen. It is well established that CT enhances Th2-mediated responses after mucosal delivery (71). Indeed, Th2 cytokine (IL-4, IL-5, IL-6, IL-10, and IL-13) mRNA responses were greatly enhanced in the lungs of mice nasally immunized with influenza Ag and CT when compared with mice given Ag alone. The expression of Th1 cytokine (IL-2 and IFN-γ) mRNAs was also increased in the lungs of mice nasally immunized with Ag combined with CT. There was about a 25-fold greater expression of IL-4 mRNA in the lung after immunization with Ag plus CT as compared with either naive mice or those immunized with Ag alone. By comparison, the increase in IFN-γ mRNA expression due to CT was relatively modest (∼5-fold). In addition, lung lymphoid cells isolated from mice immunized with influenza Ag plus CT secreted both Th1 and Th2 (IFN-γ and IL-4) cytokines after in vitro stimulation with Ag. We showed that Ag-specific CD4+ Th cells were the major source of IL-4, as their depletion eliminated IL-4 production. However, CD4+ Th cells also produced IFN-γ. CD8+ T cells, however, accounted for ∼50% of the IFN-γ produced, but CD8+ T cells were not a source of IL-4. This is in contrast to other studies demonstrating little, if any, appreciable Th1 cell activation after oral CT given as a mucosal adjuvant (71). One possibility is that Th1-type responses are transient and are not seen at later time points. Preliminary results, however, demonstrate the presence of Th1-type responses in lungs at a later time point (data not shown). Furthermore, the activation of Th1 cells is consistent with our studies (29) and those by others (72) demonstrating a significant IgG2a Ab response to Ags given nasally when CT was used as adjuvant. As the production of IgG2a is mediated by IFN-γ production, a product of Th1 cells (73), this supports the notion that nasal immunization with CT results in Th1 cell activation. As delayed-type hypersensitivity (DTH) reactions are mediated by Th1 cells (33), the generation of in vivo Th1 responses is also supported by preliminary studies demonstrating DTH responses against nasally inoculated Ag when CT is used (data not shown). Thus, our data show that CT, as anticipated, greatly enhances Th2 cell responses in the lung, but Th1 cell activation is also a significant component of the overall Th cell response.
The mechanisms through which CT enhances Th1 and Th2 cell responses in lungs are most likely linked to the development of immunopathologic reactions in the lung. These reactions are characterized by a massive infiltration of mononuclear cells around the pulmonary airways and vessels after nasal immunization using CT, but not Ag alone (28, 29). The present study demonstrated that there is an increase in the numbers of CD4+ Th cells in lungs, suggesting that they are a major component of the inflammatory infiltrate. This is similar to previous studies demonstrating an increase in B cell responses in lungs due to the recruitment of extrapulmonary lymphocytes (28). The mechanisms through which lymphocyte populations are recruited are unknown; however, our results suggest that CT induces the production of chemotactic factors, such as β-chemokines, within the lungs. β-Chemokines are cytokines that are chemotactic for mononuclear cells (60, 61, 62). Importantly, Th cell subsets differ in their responses to the β-chemokines, MIP-1α and MIP-1β (65, 74). In fact, the inclusion of CT resulted in increased levels of both MIP-1α and MIP-1β mRNA, and the levels of MIP-1β were greater than in mice immunized with Ag alone. This is consistent with previous observations showing that MIP-1α and MIP-1β are preferentially chemotactic for Th1 cells, although Th2 cells respond to MIP-1α at a lower efficiency (65). This is also supported by differences in β-chemokine cell surface receptors on Th cell subsets (75, 76). β-Chemokines may also contribute to the inflammatory infiltrate seen as β-chemokines are involved in chronic inflammatory responses, such as DTH reactions (77). However, further studies are needed to confirm the role of β-chemokines in modulating Th cell responses and associated inflammatory reactions.
Overall, the resident Th cell populations in the lungs of BALB/c mice are of a Th2 type and reflect the type of immune responses generated to nasally delivered Ag. However, we found that Th1-type cell responses can be induced in the respiratory tract during instances of intense immune stimulation, as shown after nasal immunization with Ag plus CT. In another study, we have shown a similar shift in Th cell responses in Mycoplasma respiratory disease.4The Th1 cell responses were also shown to be present in other respiratory diseases, such as those due to influenza virus (78). Although pulmonary Th1 responses may have potentially beneficial effects, they can result in inflammatory responses along the airways and vasculature in lungs. These results have not only important implications for the use of nasal immunization to prevent respiratory disease, but they provide insight to the potential mechanisms of immunity involved in infectious and immunopathologic diseases of the lung. Using this immunization model, we will be able to elucidate the mechanisms critical to the development of Th cell responses and recruitment of cells to the lungs. Future studies can then determine whether these responses can be beneficially altered by treatment with recombinant cytokines or other stimulants that elicit IgE production or cellular recruitment, leading to vaccine/adjuvant combinations, which induce appropriate protective immune responses.
We thank Leslie Tabor and Haifa Al-Khatib for their help during the performance of these studies. We also thank Dr. Maurice W. Harmon (Connaught Laboratories) for kindly providing influenza virus vaccine Ag used in these studies. We also appreciate the useful discussions and support of Drs. John Van Cott, Mark Hart, Tony Romeo, and Ronald H. Goldfarb.
↵1 This work was supported by the American Lung Association of Texas; U.S. Public Health Service Grants AI 42075 (to J.W.S.) and DC 04976, DK 44240, AI 43197, AI 18958, and P30 DK 54781; and Contract AI 65299 (to J.R.M.), DE 09837, and Contract AI 65298 (to H.K.), DE 12242 (to K.F.) from the National Institutes of Health. L.M.H. was supported in part by the Texas Advanced Technology Program under Grant 000130-0064-1999 (to J.W.S.).
↵2 Address correspondence and reprint requests to Dr. Jerry W. Simecka, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail address: jsimecka{at}hsc.unt.edu
↵3 Abbreviations used in this paper: CT, cholera toxin; β2m, β2-microglobulin; DTH, delayed-type hypersensitivity; MIP, macrophage-inflammatory protein.
↵4 H. P. Jones, L. Tabor, X. Sun, and J. W. Simecka. Depletion of CD8+ T cells exacerbates CD4+ Th cell-associated inflammatory lesions during murine mycoplasma respiratory disease. Submitted for publication.
Received May 21, 2001.
Accepted August 9, 2001.
Liang, S. C., J. W. Simecka, J. R. Lindsey, G. H. Cassell, J. K. Davis. 1999. Antibody responses after Sendai virus infection and their role in upper and lower respiratory tract disease in rats. Lab. Anim. Sci. 49: 385
Clements, M. L., R. F. Betts, E. L. Tierney, B. R. Murphy. 1986. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J. Clin. Microbiol. 24: 157
Tashiro, M., K. Tobita, J. T. Seto, R. Rott. 1989. Comparison of protective effects of serum antibody on respiratory and systemic infection of Sendai virus in mice. Arch. Virol. 107: 85
Renegar, K. B., P. A. Small, Jr. 1991. Passive transfer of local immunity to influenza virus infection by IgA antibody. J. Immunol. 146: 1972
Renegar, K. B., P. A. Small, Jr. 1991. Immunoglobulin A mediation of murine nasal anti-influenza virus immunity. J. Virol. 65: 2146
Clements, M. L., S. O'Donnell, M. M. Levine, R. M. Chanock, B. R. Murphy. 1983. Dose response of A/Alaska/6/77 (H3N2) cold-adapted reassortment vaccine virus in adult volunteers: role of local antibody in resistance to infection with vaccine virus. Infect. Immun. 40: 1044
Mazanec, M. B., C. S. Kaetzel, M. E. Lamm, D. Fletcher, J. G. Nedrud. 1992. Intracellular neutralization of virus by immunoglobulin A antibodies. Proc. Natl. Acad. Sci. USA 89: 6901
Mazanec, M. B., C. L. Coudret, D. R. Fletcher. 1995. Intracellular neutralization of influenza virus by IgA anti-HA monoclonal antibodies. J. Virol. 69: 1339
Mazanec, M. B., C. L. Coudret, T. McCool, P. Baldwin, D. R. Fletcher. 1995. Intracellular interaction between IgA antibody and viral nucleoprotein. Clin. Immunol. Immunopathol. 76: S115
Cartner, S. C., J. R. Lindsey, J. Gibbs-Erwin, G. H. Cassell, J. W. Simecka. 1998. Roles of innate and adaptive immunity in respiratory mycoplasmosis. Infect. Immun. 66: 3485
De Swert, L. F.. 1999. Risk factors for allergy. Eur. J. Pediatr. 158: 89
Sperber, K.. 1993. Asthma: an inflammatory disease. Mt. Sinai J. Med. 60: 218
Welliver, R. C., L. Duffy. 1993. The relationship of RSV-specific immunoglobulin E antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7–8 years. Pediatr. Pulmonol. 15: 19
Rossi, O. V., V. L. Kinnula, H. Tuokko, E. Huhti. 1994. Respiratory viral and Mycoplasma infections in patients hospitalized for acute asthma. Monaldi Arch. Chest Dis. 49: 107
Ostergaard, P. A.. 1977. IgA levels, bacterial carrier rate, and the development of bronchial asthma in children. Acta Pathol. Microbiol. Scand. Sect. C. 85: 187
Kraft, M., G. H. Cassell, J. E. Henson, H. Watson, J. Williamson, B. P. Marmion, C. A. Gaydos, R. J. Martin. 1998. Detection of Mycoplasma pneumoniae in the airways of adults with chronic asthma. Am. J. Respir. Crit. Care Med. 158: 998
Platts-Mills, T. A., G. Rakes, P. W. Heymann. 2000. The relevance of allergen exposure to the development of asthma in childhood. J. Allergy Clin. Immunol. 105: S503
Hughes, C. H., R. C. Jones, D. E. Wright, F. F. Dobbs. 1999. A retrospective study of the relationship between childhood asthma and respiratory infection during gestation. Clin. Exp. Allergy 29: 1378
Wahlgren, D. R., M. F. Hovell, E. O. Meltzer, S. B. Meltzer. 2000. Involuntary smoking and asthma. Curr. Opin. Pulm. Med. 6: 31
De Marco, R., F. Locatelli, J. Sunyer, P. Burney. 2000. Differences in incidence of reported asthma related to age in men and women: a retrospective analysis of the data of the European respiratory health survey. Am. J. Respir. Crit. Care Med. 162: 68
Jansen, D. F., J. P. Schouten, J. M. Vonk, B. Rijcken, W. Timens, J. Kraan, S. T. Weiss, D. S. Postma. 1999. Smoking and airway hyperresponsiveness especially in the presence of blood eosinophilia increase the risk to develop respiratory symptoms: a 25-year follow-up study in the general adult population. Am. J. Respir. Crit. Care Med. 160: 259
Elfaki, M. G., S. H. Kleven, L. H. Jin, W. L. Ragland. 1993. Protection against airsacculitis with sequential systemic and local immunization of chickens using killed Mycoplasma gallisepticum bacterin with ι carrageenan adjuvant. Vaccine 11: 311
Novak, M., Z. Moldoveanu, D. P. Schafer, J. Mestecky, R. W. Compans. 1993. Murine model for evaluation of protective immunity to influenza virus. Vaccine 11: 55
Walsh, E. E.. 1993. Mucosal immunization with a subunit respiratory syncytial virus vaccine in mice. Vaccine 11: 1135
Tamura, S., Y. Ito, H. Asanuma, Y. Hirabayashi, Y. Suzuki, T. Nagamine, C. Aizawa, T. Kurata. 1992. Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit. J. Immunol. 149: 981
Mazanec, M. B., M. E. Lamm, D. Lyn, A. Portner, J. G. Nedrud. 1992. Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract. Virus Res. 23: 1
Reuman, P. D., S. P. Keely, G. M. Schiff. 1990. Rapid recovery in mice after combined nasal/oral immunization with killed respiratory syncytial virus. J. Med. Virol. 32: 67
Hodge, L. M., M. Marinaro, H. P. Jones, J. R. McGhee, H. Kiyono, J. W. Simecka. 2001. Immunoglobulin A (IgA) responses and IgE-associated inflammation along the respiratory tract after mucosal but not systemic immunization. Infect. Immun. 69: 2328
Simecka, J., R. Jackson, H. Kiyono, J. McGhee. 2000. Mucosally-induced IgE-associated inflammation in the respiratory tract. Infect. Immun. 68: 672
Bochner, B. S., B. J. Undem, L. M. Lichtenstein. 1994. Immunological aspects of allergic asthma. Annu. Rev. Immunol. 12: 295
Simecka, J. W., S. E. Ross, G. H. Cassell, J. K. Davis. 1993. Interactions of mycoplasmas with B cells: antibody production and nonspecific effects. Clin. Infect. Dis. 17: S176
Simecka, J. W., P. Patel, J. K. Davis, S. E. Ross, P. Otwell, G. H. Cassell. 1991. Specific and nonspecific antibody responses in different segments of the respiratory tract in rats infected with Mycoplasma pulmonis. Infect. Immun. 59: 3715
Mosmann, T., R. Coffman. 1989. TH1 and TH2: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7: 145
Munk, M. E., M. Emoto. 1995. Functions of T-cell subsets and cytokines in mycobacterial infections. Eur. Respir. J. Suppl. 20: 668s
Jacobs, M., N. Brown, N. Allie, R. Gulert, B. Ryffel. 2000. Increased resistance to mycobacterial infection in the absence of interleukin-10. Immunology 100: 494
Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-γ genes. Science 259: 1739
Xu-Amano, J., H. Kiyono, R. J. Jackson, H. F. Staats, K. Fujihashi, P. D. Burrows, C. O. Elson, S. Pillai, J. R. McGhee. 1993. Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues. J. Exp. Med. 178: 1309
Xu-Amano, J., R. J. Jackson, K. Fujihashi, H. Kiyono, H. F. Staats, J. R. McGhee. 1994. Helper Th1 and Th2 cell responses following mucosal or systemic immunization with cholera toxin. Vaccine 12: 903
Simecka, J.. 1998. Mucosal immunity of the gastrointestinal tract and oral tolerance. Adv. Drug Delivery Rev. 34: 235
Finkelman, F. D., J. F. Urban, Jr, M. P. Beckmann, K. A. Schooley, J. M. Holmes, I. M. Katona. 1991. Regulation of murine in vivo IgG and IgE responses by a monoclonal anti-IL-4 receptor antibody. Int. Immunol. 3: 599
Gavett, S. H., D. J. O'Hearn, X. Li, S. K. Huang, F. D. Finkelman, M. Wills-Karp. 1995. Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice. J. Exp. Med. 182: 1527
Cohn, L., J. S. Tepper, K. Bottomly. 1998. IL-4-independent induction of airway hyperresponsiveness by Th2, but not Th1, cells. J. Immunol. 161: 3813
Foster, P. S., S. P. Hogan, A. J. Ramsay, K. I. Matthaei, I. G. Young. 1996. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med. 183: 195
Corrigan, C. J., A. B. Kay. 1992. T cells and eosinophils in the pathogenesis of asthma. Immunol. Today 13: 501
Randolph, D. A., C. J. Carruthers, S. J. Szabo, K. M. Murphy, D. D. Chaplin. 1999. Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma. J. Immunol. 162: 2375
Waris, M. E., C. Tsou, D. D. Erdman, S. R. Zaki, L. J. Anderson. 1996. Respiratory synctial virus infection in BALB/c mice previously immunized with Formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern. J. Virol. 70: 2852
Wallace, W. A., E. A. Ramage, D. Lamb, S. E. Howie. 1995. A type 2 (Th2-like) pattern of immune response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CFA). Clin. Exp. Immunol. 101: 436
Graham, M. B., V. L. Braciale, T. J. Braciale. 1994. Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection. J. Exp. Med. 180: 1273
Rousset, F., J. Robert, M. Andary, J. P. Bonnin, G. Souillet, I. Chretien, F. Briere, J. Pene, J. E. de Vries. 1991. Shifts in interleukin-4 and interferon-γ production by T cells of patients with elevated serum IgE levels and the modulatory effects of these lymphokines on spontaneous IgE synthesis. J. Allergy Clin. Immunol. 87: 58
Romagnani, S.. 1994. Regulation of the development of type 2 T-helper cells in allergy. Curr. Opin. Immunol. 6: 838
Fitch, F., M. McKisic, D. Lancki, T. Gajewski. 1993. Differential regulation of murine T lymphocyte subsets. Annu. Rev. Immunol. 11: 29
Hodges, M., K. Kingstone, W. G. Brydon, J. Sallam, A. Ferguson. 1995. Use of gut lavage fluid to measure intestinal humoral immunity, GI bleeding and protein losses in Sierra Leonean children. Adv. Exp. Med. Biol. 371B: 813
Simecka, J. W., R. B. Thorp, G. H. Cassell. 1992. Dendritic cells are present in the alveolar region of lungs from specific pathogen-free rats. Reg. Immunol. 4: 18
van Ginkel, F. W., C. Liu, J. W. Simecka, J. Y. Dong, T. Greenway, R. A. Frizzell, H. Kiyono, J. R. McGhee, D. W. Pascual. 1995. Intratracheal gene delivery with adenoviral vector induces elevated systemic IgG and mucosal IgA antibodies to adenovirus and β-galactosidase. Hum. Gene Ther. 6: 895
Kruisbeek, A. 1999. Isolation and fractionation of mononuclear cell populations. In Current Protocols in Immunology, Vol. 1. J. E. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, and W. Strober, eds. John Wiley & Sons, New York, p. 3.1.1.
Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156
Faulkner, C. B., J. W. Simecka, M. K. Davidson, J. K. Davis, T. R. Schoeb, J. R. Lindsey, M. P. Everson. 1995. Gene expression and production of tumor necrosis factor α, interleukin 1, interleukin 6, and γ interferon in C3H/HeN and C57BL/6N mice in acute Mycoplasma pulmonis disease. Infect. Immun. 63: 4084
Shakhov, A. N.. 1994. New derivative of pMUS for quantitation of mouse IL-12 (p35, p40), IL-10 and IFN-γ-R mRNAs. Eur. Cytokine Network 5: 337
Simecka, J. W.. 1999. β-chemokines are produced in lungs of mice with mycoplasma respiratory disease. Curr. Microbiol. 39: 163
Leonard, E. J., T. Yoshimura. 1990. Human monocyte chemoattractant protein-1 (MCP-1). Immunol. Today 11: 97
Schall, R. J.. 1991. Biology of the RANTES/SIS cytokine family. Cytokine 3: 165
Oppenheim, J. J., C. O. Zachariae, N. Mukaida, K. Matsushima. 1991. Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu. Rev. Immunol. 9: 617
Zingoni, A., H. Soto, J. A. Hedrick, A. Stoppacciaro, C. T. Storlazzi, F. Sinigaglia, D. D'Ambrosio, A. O'Garra, D. Robinson, M. Rocchi, et al 1998. The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J. Immunol. 161: 547
Sallusto, F., A. Lanzavecchia, C. R. Mackay. 1998. Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. Today 19: 568
Siveke, J. T., A. Hamann. 1998. T helper 1 and T helper 2 cells respond differentially to chemokines. J. Immunol. 160: 550
Locksley, R. M., F. P. Heinzel, M. D. Sadick, B. J. Holaday, K. D. Gardner. 1987. Murine cutaneous leishmaniasis: susceptibility correlates with differential expansion of helper T-cell subsets. Ann. Inst. Pasteur Immunol. 138: 744
Chatelain, R., K. Varkila, R. L. Coffman. 1992. IL-4 induces a Th2 response in Leishmania major-infected mice. J. Immunol. 148: 1182
Constant, S. L., K. S. Lee, K. Bottomly. 2000. Site of antigen delivery can influence T cell priming: pulmonary environment promotes preferential Th2-type differentiation. Eur. J. Immunol. 30: 840
Mega, J., J. R. McGhee, H. Kiyono. 1992. Cytokine- and Ig-producing T cells in mucosal effector tissues: analysis of IL-5 and IFN-γ-producing T cells, T cell receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues. J. Immunol. 148: 2030
Morawetz, R. A., L. Gabriele, L. V. Rizzo, N. Noben-Trauth, R. Kuhn, K. Rajewsky, W. Muller, T. M. Doherty, F. Finkelman, R. L. Coffman, H. C. R. Morse. 1996. Interleukin (IL)-4-independent immunoglobulin class switch to immunoglobulin (Ig)E in the mouse. J. Exp. Med. 184: 1651
Marinaro, M., H. F. Staats, T. Hiroi, R. J. Jackson, M. Coste, P. N. Boyaka, N. Okahashi, M. Yamamoto, H. Kiyono, H. Bluethmann, et al 1995. Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4. J. Immunol. 155: 4621
Reuman, P. D., S. P. Keely, G. M. Schiff. 1991. Similar subclass antibody responses after intranasal immunization with UV-inactivated RSV mixed with cholera toxin or live RSV. J. Med. Virol. 35: 192
Liang, X. P., M. E. Lamm, J. G. Nedrud. 1989. Cholera toxin as a mucosal adjuvant for respiratory antibody responses in mice. Reg. Immunol. 2: 244
Karpus, W. J., N. W. Lukacs, K. J. Kennedy, W. S. Smith, S. D. Hurst, T. A. Barrett. 1997. Differential CC chemokine-induced enhancement of T helper cell cytokine production. J. Immunol. 158: 4129
Bonecchi, R., G. Bianchi, P. Bordignon, L. D. D'Ambrosio, A. Borsatti, S. Sozzani, P. Allavena, P. Gray, A. Mantovani, F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187: 129
Hogaboam, C. M., C. L. Bone-Larson, S. Lipinski, N. W. Lukacs, S. W. Chensue, R. M. Strieter, S. L. Kunek. 1999. Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models. J. Immunol. 163: 2193
Dixon, A. E., J. B. Mandac, D. K. Madtes, P. J. Martin, J. G. Clark. 2000. Chemokine expression in Th1 cell-induced lung injury: prominence of IFN-γ-inducible chemokines. Am. J. Physiol. Lung Cell Mol. Physiol. 279: L592
Sarawar, S. R., S. R. Carding, W. Allan, A. McMickle, K. Fujihashi, H. Kiyono, J. R. McGhee, P. C. Doherty. 1993. Cytokine profiles of bronchoalveolar lavage cells from mice with influenza pneumonia: consequences of CD4+ and CD8+ T cell depletion. Reg. Immunol. 5: 142
You are going to email the following The Pulmonary Environment Promotes Th2 Cell Responses After Nasal-Pulmonary Immunization with Antigen Alone, but Th1 Responses Are Induced During Instances of Intense Immune Stimulation
Harlan P. Jones, Lisa M. Hodge, Kohtaro Fujihashi, Hiroshi Kiyono, Jerry R. McGhee, Jerry W. Simecka
The Journal of Immunology October 15, 2001, 167 (8) 4518-4526; DOI: 10.4049/jimmunol.167.8.4518
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Alder Hey Children's Hospital
Liverpool police chase: Boy, 15, arrested on suspicion of dangerous driving after dramatic high-speed pursuit
16-year-old from Kirkby, alleged to have been the passenger in the car, also held
Rob Pattinson
Queens Drive flyover looking towards Rice Lane (Image: Google Streetview)
A 15-year-old boy was arrested on suspicion of dangerous driving after a dramatic high-speed police chase across Liverpool was ended by a 'stinger' device.
The teenager, from West Derby, was taken to Alder Hey children's hospital with a minor head injury after the suspected stolen blue Volvo ploughed into railings under the Queens Drive flyover at around 12.50am following a 30-minute police pursuit.
He was arrested alongside a 16-year-old from Kirkby, alleged to have been the passenger in the car.
Eyewitnesses described the police chase, which started in Norris Green at 12.20am, as "like something from America".
Another said he saw a total of 13 police cars giving chase and said the tyres of the car being pursued "were gone".
Patrols began following the suspected stolen dark-coloured Volvo after spotting it travelling at speed on Broad Lane.
The chase continued onto Queens Drive and up to Lower Lane in Fazakerley heading for Kirkby, before later moving on to Liverpool Road towards Old Swan before the speeding car headed on to the East Lancs Road, where it was stung by police as it drove towards the Showcase cinema.
But the chase carried on, with the car then heading back to Queens Drive, where police stung it again under the flyover going towards Rice Lane.
This time the stinger was successful and the car came to a stop after colliding with railings.
A number of police cars and a police helicopter from Hawarden in north Wales were involved in the chase.
Both teenagers are being held on suspicion of aggravated vehicle taking, while the 16-year-old is also due to be quizzed on suspicion of causing £5,000 of damage to a vehicle.
The police helicopter crew later tweeted: "Assist @MerseyPolice with a high speed vehicle pursuit. Crew tracked vehicle which was eventually 'stung'. two males arrested."
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Norris Green
Old Swan

redpajama_set_name: RedPajamaCommonCrawl


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Justia Lawyer Directory Criminal Law Kansas Hiawatha Attorneys
Hiawatha Criminal Lawyers
Compare top rated Kansas attorneys serving Hiawatha.
Brad Lippert
Sabetha, KS Criminal Law Lawyer
(785) 284-3806 920 Main St
Sabetha, KS 66534
Charles Baskins
Troy, KS Criminal Law Lawyer
(785) 985-4000 137 S Main St
Troy, KS 66087
Criminal, Bankruptcy, DUI and Divorce
Richard Lake
Holton, KS Criminal Law Lawyer
Holton, KS 66436
Cheryl Marquardt
Leavenworth, KS Criminal Law Lawyer
(913) 758-1447 413 Cherokee St
Leavenworth, KS 66048
Claimed Lawyer ProfileOffers Video ConferencingQ&ABlawg SearchSocial Media
Robert C. Gigstad
10.0 (2 Peer Reviews)
(888) 387-2036 8000 Foster St.
Free ConsultationOffers Video ConferencingVideo ConfOverland Park, KS Criminal Law Attorney with 12 years of experience
Criminal, Cannabis Law, DUI and Traffic Tickets
Washburn University School of Law
Overland Park Criminal Defense Attorney Dedicated to Protecting Your Rights Law enforcement and prosecutors in the Overland Park area are committed to reducing crime and in identifying and convicting those who are engaged in criminal activities. If you have been caught in the criminal justice system, you have real reasons to be nervous. Even an innocent individual can be convicted of a crime if they are not represented by a skilled criminal lawyer that knows how to fight back. If you have been charged with a crime it is absolutely critical that you contact a skilled Overland Park criminal defense attorney from...
Michael Duma
9.0 (1 Peer Review)
(913) 782-7072 303 E. Poplar Street
Free ConsultationOffers Video ConferencingVideo ConfOlathe, KS Criminal Law Lawyer with 12 years of experience
Michael C. Duma began his legal career as a criminal prosecutor in the Wyandotte County District Attorney's Office. At his time there, he developed a skill for litigation and held an impressive conviction record. While a county prosecutor, Michael not only was successful in litigating criminal cases at the trial level, but also gained extensive experience in perfecting appeals in the Kansas Court of Appeals and Kansas Supreme Court.
Since leaving the District Attorney's Office, Michael has developed a reputation as an aggressive lawyer who skillfully advocates for his clients.
Overland Park, KS Criminal Law Attorney with 8 years of experience
(913) 735-3728 P.O. Box 13081
Overland Park, KS 66282-3081
Free ConsultationCriminal, DUI, Municipal and Traffic Tickets
I have spent my career focusing on advocating for my clients in court and before administrative bodies. The main portion of my practice focuses on Criminal Law, however, I have put my litigation experience to use for government entities, landlords, and those going through family/domestic legal issues.
Christopher Joseph
Topeka, KS Criminal Law Lawyer with 22 years of experience
(785) 234-3272 1508 SW Topeka Blvd.
Free ConsultationCriminal, Appeals and White Collar Crime
Mr. Joseph represents individuals and businesses during the investigation and prosecution of criminal charges in federal and state courts. He also represents businesses and individuals in complex civil litigation, primarily in federal court. He leads the firm's criminal and civil asset forfeiture practice groups.
Mr. Joseph received his juris doctorate in 2000 from the University of Kansas School of Law. He was invited to membership in the Order of the Coif, a national honor society for law school graduates with exceptional academic achievement. Mr. Joseph graduated summa cum laude from Wichita State University with a Bachelor of Arts degree in...
Adam Stolte
(913) 575-8823 6900 College Blvd
Free ConsultationOverland Park, KS Criminal Law Lawyer with 11 years of experience
Criminal, Appeals, DUI and Traffic Tickets
Overland Park criminal defense attorney Adam D. Stolte is a highly trained and experienced litigator committed to defending the rights of individuals accused of drunk driving and other crimes. It was during his time in the Navy working as a cryptologic technician that he realized his analytical and problem-solving skills would serve him well as an attorney. Since his graduation from the Washburn University School of Law, Mr. Stolte has his legal career fighting for the rights of individuals who have been accused of crimes in Kansas, with a focus on DUI cases. To meet with Mr. Stolte and discuss...
Topeka, KS Criminal Law Attorney with 8 years of experience
Free ConsultationCriminal, Appeals, DUI and Personal Injury
University of Kansas School of Law
Nick David opened the David Law Office in 2014 to devote his legal career to fighting for people who need someone on their side.
As a criminal defense attorney, Nick has represented clients on serious felony charges as well as misdemeanors including murder, rape, aggravated robbery, aggravated battery, burglary, criminal possession of firearms, driving under the influence (DUI), domestic violence, as well as drug offenses. On any given day, you may find Nick defending his clients throughout criminal justice process from trying cases to a jury, negotiating pleas, and preparing briefs for the Court of Appeals and Kansas Supreme Court....
Vincent Rivera
(913) 210-0844 115 E. Park St.
Free ConsultationOffers Video ConferencingVideo ConfOlathe, KS Criminal Law Attorney with 13 years of experience
Criminal, Cannabis Law, DUI and Domestic Violence
The reasons I chose to be a Kansas criminal defense lawyer are difficult to put into words. It's the feeling I get when I see somebody's car pulled over and they watch helplessly as their property is searched. I wonder: was there a legitimate reason for the stop? Did they consent to the search? When I hear on the news that a suspect is taken into custody, my first thoughts are: How did they get to be a suspect in the first place? Will they be intimidated into surrendering their rights? Do they really understand the Miranda warning? What coercive interrogation tactics...
Mr. Zach V Thomas Jr
Olathe, KS Criminal Law Lawyer with 14 years of experience
I am a criminal defense attorney serving individuals in the greater Kansas City metropolitan area in both District and Municipal courts. Prior to founding his firm, I spent years as a civil litigator; serving clients from both plaintiff's counsel and defense counsel.
My primary focus deals with defending clients charged with offenses involving domestic battery, drug and gun offenses, illegal search and seizure, and white collar crimes. I am concerned with presenting his clients with all the information so that THEY may properly weigh all of their options. There is no compulsion to take a plea to terminate a...
Paul Darrell Cramm
(913) 322-3265 7450 West 130th Street
Free ConsultationOverland Park, KS Criminal Law Attorney with 23 years of experience
Criminal, Appeals, DUI and Domestic Violence
Facing a criminal charge is ALWAYS a serious matter requiring serious defense. My Overland Park, Kansas, criminal defense practice takes every case seriously. As an experienced Johnson County Criminal Defense lawyer, I know that even a misdemeanor can have a devastating effect upon your life. In my practice, there is no such thing as a simple criminal matter.
From DUI and Drug Charges to Vehicular Homicide, Involuntary Manslaughter and Premeditated Murder, in Municipal, State or Federal Court, I provide defense of all criminal matters in Johnson County, Kansas, and the entire Kansas City Metro Area
When you are charged with a...
Brandan Davies
(913) 451-9500 7500 College Boulevard
Overland Park, KS Criminal Law Attorney with 13 years of experience
Mr. Davies has extensive experience with defending the criminally accused including DUI, felony DUI, drug possession, felony possession with intent, aggravated assault, aggravated battery, robbery, burglary, and a multitude of other misdemeanors and felony charges. Mr. Davies is dedicated to defending the rights of the accused from the multiple felon to the first time violator. Mr. Davies' trial experience and results has caused the National Trial Lawyers Association to name him as a Top 100 Trial Lawyer in 2014, 2015, and 2016. Mr. Davies has experience handling high profile cases that receive national media attention and will combat the negative...
Brian Leininger
(913) 648-7070 6900 College Blvd.
Free ConsultationCriminal, Cannabis Law, DUI and Traffic Tickets
Brian Leininger has been practicing DUI defense in Johnson County, Kansas and throughout Kansas since 2001. Prior to that he was the General Counsel for the Kansas Highway Patrol, Special Assistant Attorney General, and Assistant District Attorney in Wyandotte County. Brian is a certified practitioner of and instructor for the Standardized Field Sobriety Tests. He is the only lawyer in Kansas to have completed the training to be a Drug Recognition Expert. In addition to typical DUI cases Brian handles drug DUIs and DUI-related batteries and homicides.
Jerry L. Wallentine Jr.
Olathe, KS Criminal Law Lawyer
(913) 764-9700 130 N. Cherry St.
Free ConsultationCriminal, Civil Rights, DUI and Family
Attorney Jerry Wallentine provides representation for clients throughout Olathe who need assistance with a personal injury related matter, family law case or DUI offense. If you would like to speak with an experienced legal advocate regarding the best possible approach to your offense then you should call the firm at 816-470-0255 today.
Tarishawn D Morton
(913) 602-7288 7500 College Blvd., Suite 500
Free ConsultationCriminal and Personal Injury
Texas Southern University Thurgood Marshall School of Law
EDUCATION J.D. - Thurgood Marshall School of Law, Houston, TX M.B.A. - Colorado Techinical College, Colorado Springs, CO B.A. - Colorado State Univeristy, Pueblo, CO BAR ADMISSION Kansas Bar AWARDS National Academy Personal Injury Attorneys -Top Ten Under 40 PUBLICATIONS Criminal Justice Snapshot 2013..., ECI Legal & Social Policy (2013, Research Contribution). Focus on Justice...,ECI Legal & Social Policy (2014, Research/ Editing Contribution). "The Story of Professor Earl Lawrence Carl", Focus on Justice...,ECI Legal & Social Policy (2014, Research/ Editing Contribution). Texas Business Associations law book, (2014, PP&LLP Writing/Research Contribution). ...
John Thurston
Manhattan, KS Criminal Law Attorney
(785) 776-2000 Manhattan, KS 66502
Free ConsultationCriminal, DUI, Military and Traffic Tickets
South Texas College of Law
Carey Leonard Fleske
(913) 549-0992 8000 Foster Street
Criminal, DUI and Traffic Tickets
University of Missouri - Kansas City School of Law
Carey Fleske grew up in Great Bend, Kansas and graduated from Great Bend High School. He received his B.A. from the University of Kansas, and his J.D. from the University of Missouri in Kansas City. After he received his degree he began his legal career working for a small local firm handling criminal and civil litigation throughout the Kansas City Metro Area. After several years of working in Kansas City, Mr. Fleske had the opportunity to return to his hometown to work as an Assistant County Attorney for Barton County, Kansas. During his time as a Prosecutor he...
Mark Grover
(913) 432-1000 7270 W 98th Terr
Free ConsultationCriminal, Business, DUI and Traffic Tickets
The Grover Law Firm offers clients the best of both worlds – He is an Attorney with extensive legal expertise, knowledge and experience combined with the personal attention and focus that only a small, hardworking firm can provide.
Kate Zigtema
Lenexa, KS Criminal Law Lawyer with 16 years of experience
(913) 647-7567 8700 Monrovia
Criminal, Appeals, Domestic Violence and Traffic Tickets
Ms. Zigtema is a Kansas City native, attending Notre Dame de Sion High School. Ms. Zigtema graduated from Georgetown University, Washington, DC with a double major in International Business and Finance prior to obtaining her law degree from the University of Kansas. During her time at KU, Ms. Zigtema worked for the Johnson County District Attorney's Office gaining trial experience in the adult felony division . After graduation, Ms. Zigtema entered into private practice Ms. Zigtema's practice is primarily focused on criminal, traffic and juvenile defense at all levels, from investigation to the Kansas Supreme Court. Ms....
Christopher Kopecky
Overland Park, KS Criminal Law Lawyer with 31 years of experience
(913) 491-5599 7111 W 98th Terrace
Free ConsultationCriminal, DUI and Insurance Claims
For nearly two decades, The Traffic Lawyers of Kopecky Law, P.A. have represented thousands of clients in various traffic matters ranging from speeding tickets to complicated DUI/DWI cases. Traffic law is our life and we spend most of our day in courts all across Missouri, Kansas, and Iowa. We have several offices in the Kansas City Metro Area and one in St. Louis where we meet clients. However, we do a significant amount of work with clients completely over the internet or over the phone. Our goal is to make it as easy on you as possible. We know our...
Jerry Merrill
(913) 381-2085 7211 W 98th Ter, Suite 140
Johnson County criminal defense attorney Jerry Merrill is an experienced trial attorney who began his career as a judicial law clerk. As a former prosecutor, he has handled cases ranging from misdemeanors to high-level felonies. Jerry utilizes his insight into the way judges and prosecutors analyze cases as well as his trial experience to tirelessly pursue the best result for clients as a defense lawyer. He also substitutes for judges as a judge pro tem in the Johnson County District Court.
Lawrence, KS Criminal Law Attorney with 31 years of experience
(785) 832-2181 831 Massachusetts Street
Criminal, DUI, Traffic Tickets and White Collar Crime
Gary Stone
Kansas City, KS Criminal Law Attorney with 26 years of experience
(816) 516-8000 753 State Avenue
Criminal law attorney, Gary D. Stone, serves clients in the Kansas City, Kansas, metropolitan area and surrounding counties. As a former Assistant District Attorney who has practiced law since 1995, he has the background, skills, and proven trial record to handle your case.
Mr. Stone represents Kansas residents accused of any criminal offense in municipal and district court, and represents defendants from both Kansas and Missouri in federal court criminal and drug crimes cases. You can count on decisive and effective legal representation from Mr. Stone.
Protect Yourself—Know Your Rights
The Stone Law Office is able to provide the one-on-one consultation you need...
John Harvell
Olathe, KS Criminal Law Attorney
(913) 339-9919 108 E. Cedar Street
Free ConsultationCriminal, DUI, Domestic Violence and Traffic Tickets
Attorney John E. Harvell was born and raised in Johnson County, Kansas, where he currently practices criminal defense and serves as a judge in the city of Merriam. Mr. Harvell graduated from Shawnee Mission South High School before attending both Kansas State University and University of Kansas, graduating with honors with a degree in Sociology and Child Psychology. In 1989, he graduated from Washburn University School of Law and began his legal career trying over 25 DUI and other jury trials with the Sedgwick County District Attorneyâs office. Since that time, Attorney Harvellâs experience has been based out of Johnson County,...
Kevin Dellett
Leawood, KS Criminal Law Attorney with 27 years of experience
(913) 897-0897 4630 W. 137th Street
If you are searching for counsel to defend you in a criminal matter or to represent your interests in civil litigation, please contact me. In the 27 years I have been practicing, I have handled criminal matters ranging from minor traffic violations to serious State court felonies such as child rape and child murder, drug possession, Electronic Solicitation of a Child, Aggravated Robbery, as well as a wide range of property crimes. Defending DUI drivers in municipal and State court is a large part of my practice, ranging from first-offenders to serious felony cases. In Federal court,...
Gary L. Conwell
Topeka, KS Criminal Law Attorney with 40 years of experience
(785) 633-6767 434 S. Topeka Blvd.
Topeka, KS 66603-3152
Free ConsultationCriminal, Family, Juvenile and Traffic Tickets
Former Assistant District Attorney (Shawnee County, Kansas);
Former Assistant City Attorney (Topeka, Kansas)
104 E. Poplar Street
Free ConsultationOlathe, KS Criminal Law Lawyer with 34 years of experience
Emory University School of Law and University of Kansas School of Law
Patrick Lewis defends people accused of drug possession and sales, DUI, DWI, and violent crimes in Olathe, Johnson County, Kansas. He regularly practices in Lawrence, Douglas County, Kansas, as well as municipal courts in Johnson County, Kansas, like Overland Park, Shawnee, Lenexa, Mission and Leawood. He has tried cases ranging from traffic tickets to murder. Patrick regularly defends people against charges of sex crimes, marijuana, and domestic battery.
Stacey L. Schlimmer
(913) 341-0484 8826 Sante Fe Drive
Free ConsultationCriminal, DUI, Divorce and Domestic Violence
I am an army veteran with a former career in finance. I studied law at Washburn and before going into private practice, I worked as a prosecutor at the Johnson County District Attorney's office. I represent Kansas individuals in criminal and family law cases.
Paul Burmaster
Free ConsultationCriminal, DUI and Traffic Tickets
When you need the support of a Kansas City DUI lawyer you can trust, you should take immediate action and come to the law firm of Paul W. Burmaster P.A. This law office has earned its highly-respected reputation and is just the place for you! Attorney Burmaster received his law degree from the University of Kansas School of Law. He is a member of the Johnson County Bar Association, the Kansas Association of Criminal Defense Lawyers, and the National College for DUI Defense. Moreover, he has been admitted to the Missouri State Bar and the Kansas State Bar. He understands...
Lyndon, KS Criminal Law Lawyer with 34 years of experience
(785) 746-4172 112 East 7th Street
Lyndon, KS 66451
Criminal, Agricultural, DUI and Traffic Tickets
Attorney Michael J. Coffman is a partner at the Kansas legal office, Coffman & Campbell, LLC. With 25 years of experience under his belt and an active membership with the Kansas State Bar Association, Mr. Coffman has the knowledge and resources necessary to take on even the most complex cases. Whether you are dealing with a matter involving criminal law, estate planning or family law, you can be confident that you will have a trusted lawyer by your side. To learn more about the services Attorney Coffman may be able to provide you with, be sure to contact the firm...
Jay Norton
(913) 906-9633 9200 Indian Creek Parkway
I have primarily handled DUI cases in Johnson, Wyandotte and Douglas Counties, and all of the cities in those counties, for over 25 years.
Joshua Brunkhorst
Free ConsultationOffers Video ConferencingVideo ConfCriminal, DUI and Domestic Violence
Josh started out his career as an Assistant District Attorney in Johnson County, Kansas for nearly a decade. Throughout his career in the District Attorney's Office, Josh prosecuted hundreds of cases ranging from DUI, domestic violence offenses, sex offenses, homicides and have conducted over 30 Jury Trials. Josh has never shied away from trial and will use that same drive and experience to obtain the best possible outcome with your case.
Scott Gyllenborg
(913) 782-1400 111 South Kansas Avenue
Dwight D Alexander II
Criminal, DUI, Family and Traffic Tickets
Dwight D. Alexander, II Owner and Operator of The Alexander Law Firm, LLC Dwight is a competent and experienced attorney who created the Alexander law Firm, LLC with the client in mind. Dwight started his legal career as an intern in the Wyandotte County Unified Government Legal Department and the Kansas City, Kansas Municipal Court. During his time as an intern he crafted an idea to create a firm designed to foster a partnership with his clients to ensure that his services and his counsel created favorable results. Dwight still is apart of the Public Defenders office of...
Tyler Garretson
(913) 948-6682 105 East Park Street
Kansas City Criminal Attorney
Marc Schultz
Topeka, KS Criminal Law Lawyer
(785) 273-3900 5891 SW 29th Street
Free ConsultationCriminal, Appeals, Consumer and Juvenile
Shane Rockey
Kansas City, KS Criminal Law Lawyer with 31 years of experience
(913) 371-6063 748 Ann Ave
Criminal, DUI, Divorce and Juvenile
Mr. Heath A. Stuart
Leawood, KS Criminal Law Attorney
Free ConsultationCriminal and DUI
Doniphan County
Atchison County


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OkaySpace
Karriem Riggins Talks August Greene, J Dilla, And Wanting To Form A Supergroup With D'Angelo And Chaka Khan [Interview]
Karriem Riggins has worked with a lot of people. The Detroit-born producer and jazz drummer has crafted beats for the likes of J Dilla, Erykah Badu, The Roots, Talib Kweli, and others. Right now Riggins is focused on a project with Common and Robert Glasper called August Greene.
WATCH: Common, Robert Glasper, Karriem Riggins, And Brandy Celebrate Mississippi Activists In "Optimistic" Video
The three have all worked together prior to forming August Greene, but the group finds the three creating a full-length album together, which drops March 9. The first singles from the self-titled album — a cover of the Sounds of Blackness' "Optimistic" and the soulful "Black Kennedy," are enjoyable selections that showcase the undeniable chemistry between the three artists, celebrating both their blackness and their craft as wildly talented musicians.
Okayplayer spoke with Riggins about August Greene's self-titled debut, how the group represents black excellence, and his dream to work with D'Angelo and Chaka Khan.
Elijah C. Watson
Elijah Watson serves as Okayplayer's News & Culture Editor. When he's not writing he's listening to Sade and crying or watching My Hero Academia with his partner.
Next Music Streaming Giant Spotify Files to Go Public »
Previous « Netflix To Spend $8 Billion On Original Content In 2018
Tags: August GreeneCommonJ DillaKarriem RigginsRobert Glasper
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text:
The best iPhone/iPad USB flash drives with Lightning connectors
- Mar. 20th 2015 12:36 pm PT
There are now several companies selling USB thumb drives for iPhone and iPad since the first ones with built-in Lightning connectors debuted late last year. By connecting a small and lightweight thumb drive directly to your iOS device, you can avoid limitations of onboard storage and cloud services and instead store and stream media like music, video, and photos using the drive's additional storage.
A few things to consider when searching for the right Lightning drive: some have a battery that requires charging, companion apps for managing and streaming content from an iOS device vary in quality, and I found some have designs that make using the drives and your iOS device at the same time easier than others. Lastly, pricing varies quite a bit based on the the options below with drives ranging from 8GB to 256GB and other options offering bring-your-own storage using a micro SD card slot.
-Lightning and USB 2.0 connectors –iXpand Sync companion app for iPhone/iPad -Stream videos/music from connected drive -Auto camera roll backup -Password protected files -Needs to be charged -Cover to protect USB connector when not in use -Supports iOS, PC and Mac –16GB ($59), 32GB ($75), 64GB ($99), 128GB ($149)
Our Take: SanDisk has one of the best companion apps out of all the products on this list and some of the best prices. But the drive requires charging and has a design that forces the drive, and your iPhone attached to it, to sit awkwardly in your hand or on a table (as pictured above).
Hyper iStick
-Lightning and USB 2.0 connectors -Stream videos/music from connected drive -Auto camera roll backup –iStick companion app for iPhone/iPad -Retractable connectors -Supports iOS, PC and Mac -Colors: Silver, Black, White –8GB ($79), 16GB ($68), 32GB ($139), 64GB ($195), 128GB ($349) -(Full review)
Our Take: Hyper's iStick was originally one of the first on the market after raising over $1 million through Kickstarter last year. The product remains pricey but offers a more straightforward design that isn't obtrusive like SanDisk's design. When one connector is in use (Lightning or USB), the other end automatically retracts into the body of the drive. The app leaves a lot to be desired, however, and the prices mean you'll likely want to opt for our top pick, Leef iBridge…
Leef iBridge
-Lightning and USB 2.0 connectors –iBridge app for iPhone and iPad -Plastic, silicon and aluminum construction -Stream videos/music from connected drive -Auto camera roll backup -Capture photos directly to Leef -Cover to protect connectors when not in use -Supports iOS, PC and Mac -Colors: Black –16GB ($59), 32GB ($79), 64GB ($119) ,128GB ($199), 256GB ($399)
Our Take: Our favorite design out of all the products on the list, the Leef iBridge's wraparound design (pictured above) allows it to stay out of the way when the drive is connected and you want to continue using the device. SanDisk attempted a similar design, but iBridge did it successfully. Considering the design, pricing, a 256GB option, and solid companion app, Leef's iBridge is our top pick out of all the Lightning thumb drives on the list.
But other options are available and coming soon:
iKlips
-Lightning and USB 3.0 connectors -Companion app for iPhone/iPad -Covers to protect connectors when not in use -Stream videos/music from connected drive -Auto camera roll backup -Supports iOS, PC and Mac -Colors: Silver, Gray, Red, Gold -16GB ($55), 32GB ($69), 64GB ($125) , 128GB ($209)
Our take: It's still an about-to-launch Indiegogo project, but if you're not satisfied with any of the other options on the list, iKlips plans to outdo the competition with USB 3.0 transfer providing read/write speed of 140/20 MB/s. The other products on the list are using USB 2.0 opposite the Lightning connector. The prices listed above for iKlips represents 30% off the retail price for early bird preorders.
Amazon also has a few from little-known brands, like this one from Aizbo that accepts a micro SD card:
Aizbo i-Flash Drive with micro SD
-Lightning and USB 2.0 connectors –Companion app for iPhone/iPad -Covers to protect connectors when not in use -Stream videos/music from connected drive -Supports iOS, PC and Mac -Requires inserted SD card for storage ($28)
Options like this can be paired with inexpensive SD cards like these.
iOS is Apple's mobile operating system that runs on iPhone, iPad, and iPod touch. Historically, Apple releases a new iOS version once a year, the current version is iOS 12.
9to5Mac Reviews
All the products that the 9to5Mac team has reviewed.
app Lightning USB Flash Storage


We are down to the final 12 days of Spica wear ~ I hope forever. There is no guarantee that Sara's surgery will forever fix her dislocated left hip. In fact, our surgeon has told us repeatedly how lucky she will be if this is the only surgical intervention she will need throughout her life. I hate the thought of her going through this again a few years down the road. But I'm hanging on to hope because that's all I have.
Very kind strangers have made this "ordeal" much more manageable. People I have never met sent us supplies we would need post surgery – out of the kindness of their hearts. One family sent us babylegs, tape, and moleskin. Another family sent us a cast cooler.
The kindness of strangers is a powerful thing, in deed. It makes the world smaller and a bit more hopeful. My family (spurred on my generous 8 year old) passed out envelopes of cash on Christmas Eve morning in a low income area of our city. We did not have to go looking for the ideal recipients. They were simply there. My faith tells me that when you have hope to give and don't hold back, the person who needs it will receive it. We have decided to make this outing a yearly tradition. Sending love and hope to your family as you get through the last 12 days of Spica wear.
What a sweet, kind, thoughtful gesture - handing out money on Christmas! I've found that even giving someone the pathetic little quarter in my Aldi cart can really brighten a day. I can't imagine getting an envelope T of cash! Good for you!! Thank you for sharing.
Thanks, Monique! I hope everything goes very well with Chloe, too!
Extending a kind and helpful hand to others is the best way to create community around you and your family. When we first moved into our neighborhood I was childless and not working due to health issues. I tutored many children in our neighborhood free of charge. In doing so I made many wonderful friends. They would often send over extra food so I wouldn't have to cook dinner after tutoring their children. One uncle re-did our kitchen cabinets for free, we just had to buy the materials. Now I have an active toddler and don't tutor regularly although I help with special projects when asked. I still benefit from warm relationships and now these families will occasionally care for my son when I have an appointment. For no charge, of course! I could have earned a lot of money by tutoring for a fee, but I think that by gaining community I came out much richer in the end!


text:
Q: Bloqueio na porta 80 do apache Está sendo desenvolvido dentro da empresa uma aplicação para um controle, porém quando tento utilizar o apache em outras máquinas para que tenha acesso retorna um bloqueio

Forbidden
You don't have permission to access /backup on this server.
  Apache/2.4.18 (Win64) PHP/5.6.19 Server at 192.168.123.22 Port 80

Obs: Se colocar na 8080 ele reconhece normalmente, porém precisa ser a 80, estamos utilizando a ultima versão do apache.

A: A resposta Forbidden significa que você está acessando sim o Apache, mas a página não é autorizada, quero dizer isto não é um problema com a porta, é apenas um problema de permissões.
WampServer
Provavelmente está usando WampServer, se for este o caso basta clicar na opção Colocar online (ou Put online se estiver em inglês) assim:

Outros que não sejam WampServer
Se estiver usando outro tipo de servidor que usa Apache você deve editar o httpd.conf
Ele deve estar algo como (use o Ctrl+F para procurar algo semelhante):
<Directory "d:/wamp/www">
     Order Deny,Allow
     Deny from all
     Allow from 127.0.0.1
     Allow from ::1
     Allow from localhost
</Directory>

Neste caso bloqueia a todos e libera apenas os 3 últimos, então basta adicionar o que vai liberar, no caso o ip é 192.168.123.22 (se for fixo), então seria algo como:
<Directory "c:/wamp/www">
     Order Deny,Allow
     Deny from all
     Allow from 127.0.0.1
     Allow from ::1
     Allow from localhost
     Allow from 192.168.123.22 #Ip da sua maquina
</Directory>

E então reiniciei o Apache, reinicie o computador (as vezes só basta fazer logoff no Windows)
Ou remova tudo (faça um backup do httpd.conf primeiro, antes de editar qualquer coisa)
E então reiniciei o Apache.
Você disse que só ocorre com porta 80, isso é porque as configurações pode estar no VirtualHost:
<VirtualHost *:80>
    ServerName localhost
    DocumentRoot c:/wamp/www
    <Directory  "c:/wamp/www/">
        Options +Indexes +FollowSymLinks +MultiViews
        AllowOverride All
        Require local
    </Directory>
</VirtualHost>

O Require local pode ser o motivo do bloqueio também (lembre-se antes de remover qualquer coisa faça um backup).
Documentação: https://httpd.apache.org/docs/2.4/howto/access.html

A: Abra o command prompt em modo administrativo e execute:
netstat -np TCP | find ":80"

Os IPs a esquerda é o seu IP local. O comando acima retornará dezenas ou centenas de resultados se estiver navegando ou com algo conectado a internet.
Um jeito rápido de filtrar e obter o que interessa é colocar o seu IP no comando
Exemplo, se o seu IP é 192.168.1.10
netstat -abnop TCP | find "192.168.1.10:80"

Isso retornará vazio se não houver nada.
Caso tenha algo, retornará:
TCP    192.xx    192.xx    ESTABLISHED    4444

O último número é o PID do processo em questão.
Para descobrir o nome do processo:
tasklist /fi "pid eq 4444"

Retorna:
Image Name      PID      Session Name    Session#     Mem Usage
processo.exe    4444     nome_sessao     2            10k

Assim você obtém o nome do programa que está ocupando a porta que deseja, no caso a porta 80.
Dois programas não podem ocupar a mesma porta.
Caso queira o Apache nessa porta que está em uso, deve interromper o outro programa ou mudá-lo para outra porta.
Obs: A sua questão ainda parece confusa porque a mensagem de erro para quando a porta está em uso é diferente da mensagem que está na pergunta.
A mensagem é normalmente relacionada com permissão de leitura e escrita de um determinado local físico.

redpajama_set_name: RedPajamaStackExchange


text:
Q: By RFC 1928, what should SOCKS5 client do when receive UDP reply message which ATYP is 0x03 (domain)? According to RFC 1928, we know the UDP request and reply header as follow:
 +----+------+------+----------+----------+----------+
 |RSV | FRAG | ATYP | DST.ADDR | DST.PORT |   DATA   |
 +----+------+------+----------+----------+----------+
 | 2  |  1   |  1   | Variable |    2     | Variable |
 +----+------+------+----------+----------+----------+

 ATYP address type of following addresses:
   o  IP V4 address: X'01'
   o  DOMAINNAME: X'03'
   o  IP V6 address: X'04'

In reply scenario, when the ATYP is 0x03 (domain), how can the client know the real IP address of the remote destination?

A: When receiving a datagram from a remote host, the ATYP should not be X'03'. It should always be either X'01' or X'04' to carry the IP address of the reply sender. But, if for some reason, the UDP relay decides to set ATYP to X'03', it means the IP is being hidden, so there is no way for the receiving client to know what the sender's IP is.
When sending a datagram to a remote host, the ATYP may be X'03', if the sender wishes the UDP relay to use its own DNS resolution to determine the remote IP to send to.


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Dhaka, May 1: BNP chairperson Khaleda Zia on Sunday accused the government of plundering public money in the name of development.
The former prime minister also alleged that the ruling Awami League (AL) has siphoned off Tk 30,000 crore over the last seven years.
The BNP chief came up with the allegation while addressing a workers' rally at Suhrawardy Udyan in the city.
She demanded of the government either release Shafik Rehman and Amar Desh editor Mahmudur Rahman or arrest Joy and grill him over the laundered Tk 2,500 crore.
Khaleda said journalist Shafik Rehman was arrested and put on remand as he collected some information about Prime Minister's son Sajeeb Wazed Joy's involvement in siphoning off Tk 2,500 crore.
"The ruling party often talks about development…they've plundered more public money than what they spent on development. Now they're taking mega projects in the name of development and looting huge money. Awami League has plundered Tk 30,000 crore over the last seven years," she said.
She urged to speed up movements against the Awami League (AL) government taking lessons from the May Day.
"Verify your popularity arranging immediate general election under the nonparty government," she said pointing the ruling AL.
There can be no 'fair election' under the present Election Commission, she said. "We will not allow another general election under this government," she added.
BNP's workers' front Jatiyatabadi Sramik Dal arranged the programme, marking the May Day.