extfieldsofstudy: Chemistry Biology

s2fieldsofstudy: Biology Chemistry Medicine

text:
IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM: To examine the pathway related to the IL-1β-induced activation of mitogen-activated protein (MAP) kinases in cat esophageal smooth muscle cells.

METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1β. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases.

RESULTS: In the primary cultured cells, although IL-1β failed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP kinase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively. The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level. Phosphokinase C (PKC) was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity. In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds.

CONCLUSION: Based on these results, IL-1β-induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1β.

INTRODUCTION
Esophageal reflux is a common condition that affects both children and adults. If left untreated, it can result in chronic esophagitis, aspiration pneumonia, esophageal strictures and Barrett's esophagus, which is a premalignant condition [1] . Reflux esophagitis (RE) is a multifactorial disease that may depend on the relaxation of the transient lower esophageal sphincter (LES), the speed of esophageal clearance, the mucosal resistance and other factors, and is often associated with the LES pressure [2] . Cytokines exhibit potent chemotactic activity toward different populations of leukocytes, and are essentially involved in the induction of acute and chronic inflammatory reactions [3,4] . The selective infiltration of various leukocyte subsets into the esophagus indicates the participation of chemokines in the immune and inflammatory processes of RE.
Interleukin-1 (IL-1) has several biological effects on many cell types, and plays a key role in regulating the inflammatory reaction [5] . The IL-1 family is an important part of the innate immune system, which regulates functions of the adaptive immune system. In the presence of excess IL-1, inflammatory and autoimmune diseases can develop in many organs, including the joints, lungs, gastrointestinal track, central nervous system, or blood vessels. Stimulation of the cells by IL-1 initiates the transcription of many pro-inflammatory genes, including IL-6 and IL-8. The levels of the inflammatory genes are higher in the esophageal mucosa of those patients with RE [6] . IL-1 has its own identical biological functions and it is induced by gastric ulceration in rats [7,8] . Increased mucosal levels of IL-1 are constantly observed during acute and chronic intestinal inflammation in human beings [9,10] and animals [11,12] . The immunoneutralization of the IL-1 activity has been reported to greatly reduce the severity of colitis in a murine model of the disease, suggesting that this cytokine plays an important role in initiating inflammation [12] . The IL-1 mRNA and protein were strongly expressed in the gastric mucosa of gastritis and ulcer patients [13,14] . Therefore, it is suggested that the pro-inflammatory effects of IL-1 are due to the induction of COX-2 gene expression in many tissues, including mucous [14] . However, there are few reports about the effect on the smooth muscle.
In mammalian cells, there have been at least three different subfamilies of mitogen-activated protein (MAP) kinases reported. They include the extracellular signalregulated kinases (ERKs, p44/42 MAP kinase), c-Jun N-terminal kinase (JNK), and p38. These kinases are activated by distinct upstream MAP kinases (MEKs), which phosphorylate the residues within a tripeptide motif (Thr-X-Tyr) on the MAP. Once activated, MAP kinases, in turn, phosphorylate a variety of intracellular substrates, including certain transcription factors [15] . I L -1 β is known to activate all three MAP kinase subfamilies in human airway smooth muscle cells. IL-1β stimulation has been associated with several MAP kinases cascades involved in the transmission of a signal from the cell surface to the nucleus. Although these pathways have not been demonstrated to be involved in the IL-1β-mediated effects on the esophageal smooth muscle, p38 and ERK have been shown to contribute to such effects in human [16] or canine airway [17] or myometrial [18] smooth muscle. Therefore, in this study, we examined the mechanism associated with IL-1β-induced MAP kinases activation in cat esophageal smooth muscle cells.

Preparation of feline esophageal smooth muscle tissue and cell culture
Adult cats of either gender, weighing between 2.5 and 3.5 kg, were used in this study. The cats were anesthetized with ketamine (50 mg/mL per kg) and the abdomen was then opened with a midline incision. The esophagus and stomach were excised together, cleaned and maintained in Krebs buffer with the following composition (mmol/L): NaCl 116.6, NaHCO3 21.9, NaH2PO4 1.2, KCl 3.4, CaCl2 2.5, glucose 5.4 and MgCl2 1.2. The esophagus and stomach were opened along the lesser curvature. The location of the squamocolumnar junction was identified and the mucosa was peeled off. The high-pressure zone was identified by a visible thickening of the circular muscle layer in conjunction with the squamocolumnar junction and immediately proximal to the sling fibers of the stomach. It has been shown that a 5-to 8-mm band of tissue coinciding with the thickened area constitutes the LES, and has distinct in vivo characteristics in an organ bath or as single cells after enzymatic digestion [19,20] .
After opening the esophagus and stomach and identifying the LES, the mucosa and submucosal connective tissues were removed via a sharp dissection. The LES was excised and a 3-to 5-mm wide strip at the junction of the LES and esophagus was discarded in order to avoid potential overlap. The circular muscle layer from the esophagus was cut into 0.5-mm-thick slices using a Stadie Riggs tissue slicer (Tomas Scientific Apparatus, Philadelphia, PA, USA). The last slices containing the myenteric plexus, the longitudinal muscle and the serosa were discarded, and the remaining slices were then cut into 2 mm × 2 mm tissue squares by hand.
T he sliced tissue was then placed into DMEM supplemented with 500 mL/L FBS containing 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mL amphotericin B and incubated at 37 ℃ in a humidified atmosphere containing 50 mL/L CO2 and 950 mL/L air. On the following day, fresh DMEM containing 1 0 0 mL/L FBS was added. Ten days later, the tissue explants were removed and the medium was exchanged with fresh DMEM containing 100 mL/L FBS. After the cells reached confluence, the cells were detached with 10 g/L trypsin-EDTA in HBSS with sodium bicarbonate. The cells were then counted, seeded at 1×10 6 cells/mL on 100-mm culture dishes, and maintained in DMEM containing 100 mL/L FBS. The medium was changed every 48 h until the cells reached confluence. The experiments were performed on the cells of passage 2.
In order to characterize the isolated and cultured esophageal smooth muscle cells as well as to exclude contamination by epithelial cells and fibroblasts, the cells were identified using an indirect immunofluorescent staining method with a monoclonal antibody of a light chain myosin [21] . More than 95% of the cell preparation was found to be composed of smooth muscle cells.

Preparation of cell extracts and Western blot analysis
For the animal esophagitis model, esophageal smooth For the IL-1β treatment experiments, the cells were plated in six-well culture plates and made quiescent at confluence by incubating them in serum-free DMEM for 24 h. The growth-arrested esophageal smooth muscle cells were incubated with or without IL-1β at 37 °C at various times. When the inhibitors were used, they were added 1 h before applying the IL-1β according to the inhibitors, with the exception that the cells were pretreated with the pertussis toxin 24 h prior to IL-1β. After incubation, the cells were rapidly washed with ice-cold PBS and lysed for 5 min in an ice-cold lysis buffer containing 25 mmol/L Tris-HCl (pH 7.4), 25 mmol/L NaCl, 25 mmol/L NaF, 25 mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 2.5 mmol/L EDTA, 2.5 mmol/L EGTA, 0.5 g/L Triton X-100, 5 g/L SDS, 0.5 g/L deoxycholate, 0.5 g/L NP-40, 5 μg/mL leupeptin, 5 μg/mL aprotinin, and 1 mmol/L PMSF. After incubation, the lysates were scrapped and collected by centrifugation at 45 000 g for 1 h at 4 °C to yield the whole cell extract.
Equal amounts of the protein from each sample were resolved on a SDS-polyacrylamide gel by electrophoresis. Prestained molecular mass markers were also run in an adjacent lane to deter mine the molecular mass. The separated proteins were transferred to a 0.45-μm nitrocellulose membrane in 25 mmol/L Tris (pH 8.3), 192 mmol/L glycine, and 100 mL/L methanol using a power supply (Power Pac 1000, BioRad, Melville, NY, USA). The membranes were incubated in a PBS buffer containing 50 g/L non-fat dry milk for 1 h at room temperature in order to block the nonspecific binding. After washing thrice with PBS, the blots were incubated with the primary antibodies in a PBS solution containing 1 g/L BSA at 4 °C overnight. The membranes were washed using PBS containing 0.5 g/L Tween 20 and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:1 000 dilution) for 1 h. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham). The total MAP kinase expression level was determined by subsequently stripping the same blot with a stripping buffer and reproving the blot with p44/42 MAP kinase antibodies. Developed films from ECL were scanned and analyzed densitometrically using Scion Image.

Protein determination
The protein concentration of the supernatant in each reaction vial was measured spectrophotometrically at a wavelength of 595 nm using a BioRad assay (BioRad Chemical Division, Richmond, CA, USA).

Statistical analysis
The data were expressed as mean ± SE of three separate experiments, and the statistical differences between means were determined by using Student's t test. A P < 0.05 was considered statistically significant.

RESULTS
The treatment of the esophageal smooth muscle cells (ESMC) with IL-1β did not increase the COX-2 expression level ( Figure 1). In other cell types such as canine tracheal smooth muscle cells [22] , human gastric cancer cells [23] , and human myometrial smooth muscle cells [18] , it has been reported that IL-1β exerts its biological effects partly by inducing COX-2 expression. However, the cells treated with IL-1β at a concentration of 0.5-50 ng/mL for 18 h did not show an increased COX-1 and COX-2 expression levels ( Figure 1A). In addition, when treated with 25 ng/mL IL-1β for 15 min to 24 h, the COX expression level in the cat ESMC was not altered ( Figure 1B). These results corresponded to previous results showing that an experimentally induced rat esophagitis did not cause an increase in the COX-2 level, suggesting that COX-2 might not mediate esophagitis (unpublished data).
It is also known that IL-1β mediates inflammation, at least in part, by inducing the expression of inducible nitric oxide synthase (iNOS) [7] , with the concomitant enhanced synthesis of NO, which is a critical effector molecule, in rat pulmonary artery smooth muscle cells. The cat ESMCs were treated with IL-1β (25 ng/mL) for 18 h and the iNOS level was determined by using Western blotting analysis ( Figures 1C and 1D). This treatment did not increase the iNOS expression level, suggesting that iNOS, like COX-2, might not be a mediator of the cellular response to IL-1β in ESMC.
Although there was no change in the COX and iNOS levels, which is similar to other reports, IL-1β activated p44/42 MAP kinase with increasing concentration (Figure 2). After a treatment with 0.5-50 ng/mL IL-1β for 3 h, p44/42 MAP kinase was phosphorylated at concentrations above 10 ng/mL. Densitometric assessments demonstrated that the exposure of ESMCs to 25 ng/mL IL-1β for 3 h significantly increased the level of activation of p44/42 MAP kinase by 2.9 ± 0.1 times over the level of control samples ( Figure 2A). Furthermore, IL-1β (25 ng/mL) produced a time-dependent increase in p44/42 MAP kinase phosphorylation ( Figure 2B). The effect of IL-1β on p44/42 MAP kinase was observed within 30 min. The maximum increase (2.6 ± 0.1-fold increase over the control) in this response was observed within 3 h, and a gradual decrease to the basal level within 18 h was observed. Based on the concentrationresponse and time-course data, the ESMCs were exposed to I L -1 β at a final concentration of 25 ng/mL for 3 h in later experiments, aiming at identifying the signaling pathway of p44/42 MAP kinase activation. Similarly, p38 MAP kinases were activated by IL-1β in the cat ESMCs. As shown in Figure 2A, an 18-h IL-1β treatment caused phosphorylation of p38 MAP kinase in a concentrationdependent manner. A clear increase (1.9 ± 0.1-fold) was observed at concentrations above 10 ng/mL IL-1β. The time course of p38 MAP kinase phosphorylation indicated that the IL-1β increased the activity within 12 h, which was maintained up to 24 h (1.5 ± 0.04-fold increase over the control). Therefore, even though IL-1β activated p38 MAP kinase as well as p44/42 MAP kinase, a difference in the activated time of the two MAP kinases was also observed. This suggests that IL-1β can evoke these cellular responses via at least two pathways, involving p38 MAP kinase and p44/42 MAP kinase, in the cat ESMCs.
In order to determine if the effects of IL-1β on p44/42 and p38 MAP kinases were mediated through the activation of a receptor coupled to the pertussis toxin (PTX)-sensitive G protein, the ESMCs were pretreated with 100 ng/mL PTX for 24 h, followed by stimulation with IL-1β for 3 and 18 h, respectively ( Figure 3A). Previous studies showed that various types of G proteins, such as Gs, Gq, Go and Gi1-3, could be detected in the cat ESMCs [24] . As shown in Figure 3A, pretreatment of these cells with PTX caused a slight attenuation of p44/42 phosphorylation, indicating that IL-1β exerts its effects through the receptor-coupled Gi protein. However, p38 MAP kinase phosphorylation was maintained without being mediated by the Gi protein. In order to determine the involvement of adenylate cyclase (AC) in the activation of MAP kinases, the cells were pretreated with forskolin 24 h before IL-1β in order to activate the AC (Figure 2A). AC had little effect on p44/42 MAP kinase activation, but was found to decrease the phosphorylated forms of p38 MAP kinase. Therefore, it is believed that IL-1β-induced p38 MAP kinase activation is negatively regulated by cAMP.
Recently, it has been proposed that IL-1β causes the activation of tyrosine kinase and regulates protein tyrosine phosphorylation [22] . There are two classic tyrosine kinases, namely, receptor and nonreceptor tyrosine kinase. The receptor tyrosine kinases are actually the growth factor receptors located in the inner side of the cytoplasmic membrane and undergo dimerization and autophosphorylation upon activation. In this study, we examined the possibility that the observed increase in the level of MAP kinases phosphorylation after an IL-1β treatment might be mediated by tyrosine kinase ( Figure  3B). Therefore, the cells were pretreated with genistein for 1 h prior to adding IL-1β. This resulted in a decrease in IL-1β-induced p44/42 MAP kinase activation. In addition, tyrphostin 51, which is a member of the genistein family of tyrosine kinase inhibitors and is specific to the receptor tyrosine kinase, significantly decreased the level of p44/42 MAP kinase phosphorylation, suggesting that tyrosine kinase might participate in these responses to IL-1β.
The involvement of phospholipase in the IL-1βinduced p44/42 MAP kinase activation was examined using a phospholipase C (PLC) inhibitor (neomycin), a PLA2 inhibitor (DEDA), and a PLD inhibitor (ρCMB). As shown in Figure 4A, pretreatment of the ESMCs with neomycin attenuated the p44/42 MAP kinase activation. DEDA also slightly decreased the band density, but could not reach statistical significance. In addition, pretreatment of the ESMCs with ρCMB did not decrease the level of p44/42 MAP kinase activation induced by IL-1β, suggesting that IL-1β-stimulated p44/42 activation in ESMC might be mediated via the activation of PLC. In this study, we therefore investigated whether or not    Figure  4B), which reduced the level of p44/42 MAP kinase phosphorylation induced by IL-1β. It was assumed that there was no relationship between p38 MAP kinase phosphorylation and the phospholipases ( Figure 4A). As the pretreatment with the phospholipase inhibitors had no effect on phospho-p38 MAP kinase band density, the PKC inhibitors did not decrease the level of activation by IL-1β ( Figure 4B). Overall, IL-1β might activate p38 MAP kinase in cat ESMCs via a pathway other than the phospholipaseand PKC-associated pathway. The relationship between the activation of the two MAP kinases was identified after treating the ESMCs with either 10 μmol/L PD98059 (a synthetic inhibitor of MEK1/2 activation) or 30 μmol/L SB202190 (a p38 MAP kinase inhibitor) for 1 h ( Figure 5A). PD98059 resulted in inhibition of the IL-1β-induced p44/42 MAP kinase activation. However, SB202190 had little effect, indicating that p44/42 MAP kinase activation in ESMCs was mediated by the upstream kinase MEK, which was not affected by p38 MAP kinase. SB202190 markedly decreased the level of p38 MAP kinase phosphorylation to almost the basal control level, whereas PD98059 had little effects on this activation. This means that the IL-1β-induced p38 MAP kinase and p44/42 MAP kinase activation might be accomplished by independent pathways.
Although IL-1β did not enhance the COX-2 and iNOS expression levels, the participation of COX and NOS in the activation of p44/42 MAP kinase by IL-1β is still unclear. Therefore, the cells were treated with a COX inhibitor, indomethacin, and a NOS inhibitor, L-NAME, for 1 h before IL-1β stimulation in order to determine whether COX and NOS play a role in the activation ( Figure  5B). The increase in the phosphorylated forms of MAP kinases was maintained even with the pretreatment of inhibitors. This suggested that COX and NOS played no role in IL-1β-induced MAP kinases activation.

DISCUSSION
It is evident that the proinflammatory cytokines and chemokines play an important role in the pathogenesis of various inflammator y conditions [3,4,25] . Recently, it was reported that there was a significantly higher expression level of IL-8 mRNA in biopsy specimens obtained from RE patients than that from non-inflamed samples, which was determined by competitive reverse transcriptase polymerase chain reaction [26] . Furthermore, in addition to IL-8, the MCP-1 and RANTES levels were also significantly higher in esophagitis patients. In that report, although there were no significant differences in the mucosal IL-1β levels between the RE samples and the control group, the tissue IL-1β levels correlated significantly with the level of IL-8 production [6] . Cytokines, such as IL-1β and TNF-α, act locally and systemically to recruit and activate the target cells that can produce additional cytokines. IL-1 induces the gene expression of neutrophils and monocyte chemotactic cytokines, such as IL-8, IL-9, and macrophage inflammatory proteins that in turn stimulate migration and degranulation of neutrophils in vivo [27] . The surgically induced esophagitis rats resulted in an approximately two-fold increase in the level of TNF-α and IL-1β production [28] .
Previous studies demonstrated that COX-2 and iNOS were the effectors of the IL-1β-induced cellular responses, products of which were PGE2 and NO, respectively. In our previous study, although iNOS expression and nitrite levels in the tissue were higher after inducing esophagitis in rats (unpublished data), the COX-2 level was unaffected by each IL-1β concentration ranged from 0.5 to 50 ng/mL. In addition, the IL-1β treatment by 24 h failed to increase the iNOS concentration in the cultured cat ESMCs. Therefore, IL-1β did not play a direct role in the inflammatory process in the esophagus, suggesting that IL-1β might be related to the induction of other cytokines, which would be an immediate cause of the inflammatory cellular response, such as COX-2 and iNOS, in cat ESMC.  IL-1β plays its own role in the cellular signaling components in the cat ESMC. The MAP kinase pathway has been suggested to be a mechanism by which various signals are transduced from the cell surface to the nucleus in response to a variety of different stimuli. These proteins participate in several intracellular processes by further inducing the level of phosphorylation of various intracellular substrates, such as other protein kinases and transcription factors. This signaling mechanism is believed to control a wide spectrum of cellular physiological and pathophysiological processes including cell growth, differentiation, and the stress response [29] . The treatment of the ESMC with > 10 ng/mL IL-1β resulted in the activation of p44/42 MAP kinase, which was generated by 30 min and reached a maximum within 3 h, with a subsequent decrease. A similar pattern was observed with p38 phosphorylation, which was also concentrationdependent above 10 ng/mL. This increase was observed after 12 h, suggesting that p44/42 MAP kinase might participate in the early phase response to IL-1β, and p38 MAP kinase might be a mediator of the late phase IL-1β effects.
In order to determine whether IL-1β interacts with the G proteins, the requirement of the G protein was evaluated using ESMC pretreated with the pertussis toxin (PTX). PTX has been shown to inhibit the intrinsic GTPase activity of the Gα subunit by the ADPribosylation of specific residues. A previous report demonstrated that Gq, G11, Gi1, Gi2, and Gi3 were present in the esophagus and LES [24] . In this study, the IL-1β-induced activation of p44/42 MAP kinase was affected by the PTX pretreatment, suggesting the involvement of a PTX-sensitive G protein in these processes. However, PTX had little effect on the IL-1β-induced activation of p38 MAP kinase. This inhibitory effect of the toxin might result from the increases in the cyclic AMP (cAMP) level via the abrogation of the Gi protein in ESMC. A previous report demonstrated that IL-1β activated AC in vascular smooth muscle via COX-2 [30] . Therefore, a cAMP elevating agent, forskolin, was used to activate adenylate cyclase in order to evaluate its role in IL-1β-induced responses. Forskolin failed to attenuate the IL-1β-induced p44/42 MAP kinase activation, excluding the involvement of elevated cAMP. In contrast, the cAMP concentration affected the level of p38 MAP kinase phosphorylation, suggesting that the delayed activation of p38 MAP kinase is due to the elevation of cAMP by IL-1β.
It is well known that IL-1β triggers the activation of PI-PLC and PC-PLC in U937 cells and tracheal smooth muscle cells to generate DAG and IP3 [31,32] . DAG and IP3 activate PKC and release Ca 2+ from the intracellular stores, respectively, and may play an important role in regulating the cellular functions in several cell types [33] . PKC is a major component in the kinase cascade that is initiated by ligand attachment to both the G protein-coupled receptors and the receptors possessing intrinsic tyrosine kinase activity. Different PKC isozymes appeared to mediate the LES tone as well as the phasic contraction of the ESMC [34] . In order to confirm the participation of phospholipase in the IL-1β-induced activation of MAP kinases, the cells were pretreated with inhibitors of each phospholipase.
Neomycin markedly decreased the activity of p44/42 MAP kinase, but failed to reduce the activity of p38 MAP kinase. Therefore, p44/42 MAP kinase and p38 MAP kinase are on a different IL-1β signaling pathway at least downstream from the receptors. The regulatory mechanisms involved in IL-1β-induced MAP kinase activation by PKC were further investigated. These results showed that pretreatment with the PKC inhibitors attenuated the IL-1β-induced p44/42 MAP kinase activation in the ESMC. These results were consistent with those showing that cytokine activated the PLC-coupling signaling pathways in the human histiocytic lymphoma cell line, U937 [31] . Despite the relationship between p44/42 MAP kinase activation by IL-1β and PKC, the PKC inhibitors did not exert any inhibitory effects on p38 MAP kinase activation.
The implication of a tyrosine kinase in the activation MAP kinases by IL-1β was investigated using tyrosine kinase inhibitors, genistein and tyrphostin 51. Genistein and tyr phostin 51 inhibited p44/42 MAP kinase phosphorylation, indicating the involvement of tyrosine kinase in this response. In contrast, these inhibitors did not block the phosphorylation of p38 MAP kinase by IL-1β.
The activation of p44/42 MAP kinase requires both tyrosine and threonine phosphorylation as a result of the dual specificity MEK1/2. PD98059, a synthetic and highly specific MEK1/2 inhibitor, has been shown to inhibit p44/42 MAP kinase activation by several stimuli [35] . In this study, a PD98059 pretreatment attenuated the p44/42 MAP kinase activation, leaving p38 MAP kinase phosphorylated. In addition, IL-1β has been shown to phosphorylate p38 MAP kinase, which appears to be distinct from p44/42 MAP kinase in several cell types [36,37] . SB202190 inhibited the IL-1β-induced p38 MAP kinase activation, but did not show any inhibitory effect on p44/42 MAP kinase activation. Overall, the activation of p44/42 MAP kinase and p38 MAP kinase appears to involve independent pathways.
The absence of a role of COX and iNOS in these pathways was confirmed by their respective inhibitors, indomethacin and L-NAME. Indomethacin plays a role in preventing COX-2 from producing prostaglandin, and L-NAME blocks NO synthesis by NOS. The IL-1βinduced activity of p44/42 MAP kinase and p38 MAP kinase remained unchanged even after the indomethacin and L-NAME treatment, thereby excluding the role of prostaglandin and NO in this responses.
In conclusion, IL-1β, which is believed to act as a proinflammatory mediator by evoking other cytokines in the esophagitis, can induce p44/42 MAP kinase and p38 MAP kinase activation. The pathway related to p44/42 MAP kinase is composed of a PTX-sensitive G protein, tyrosine kinase, PLC and PKC. These components are believed to play a role in the early cellular responses via IL-1β-induced p44/42 activation, but have little effect on p38 MAP kinase phosphorylation, which is generated later and is regulated negatively by cAMP.


extfieldsofstudy: Biology Medicine

text:
Myeloperoxidase Promoter Polymorphism −463G Is Associated With More Severe Clinical Expression of Cystic Fibrosis Pulmonary Disease

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the −463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.

INTRODUCTION
Cystic fibrosis (CF) is a lethal autosomal recessive disorder which is caused by mutation of the cystic fibrosis transmembrane regulator (CFTR) gene [1]. Although CF is a multiorgan disease, most of the morbidity and the mortality are due to progressive pulmonary disease with lung parenchymal destruction and chronic pulmonary bacterial colonization [2,3]. There is a hypersecretion and a massive influx of neutrophils within the airways which mediate deleterious effects [4,5]. Moreover, the intensity of the airway inflammation is directly correlated with the clinical status as evidenced by a close correlation between respiratory scores and neutrophilderived proteinases [6]. The airway inflammation typical of CF relies thus on the paradox of misdirected neutrophil microbicidal activity resulting in an exacerbation of neutrophilmediated tissue damage and a concomitant failure in the antimicrobial system, since CF patients can become chronically infected with Pseudomonas aeruginosa (P aeruginosa) [7,8]. Activation of the neutrophil results in the oxidative burst which occurs at the level of NADPH oxidase leading to the generation of superoxide anion which can dismutate into hydrogen peroxide (H 2 O 2 ) [9,10]. Myeloperoxidase (MPO), an enzyme contained in azurophil granules, catalyzes the H 2 O 2 -dependent oxidation of chloride (Cl − ), which yields hypochlorous acid (HOCl) [11]. HOCl is a potent microbicidal agent and a toxic chlorinating oxidant. MPO released from activated neutrophils can also damage bystander cells at inflammatory sites. Airway secretions from CF patients contained high concentrations of active MPO [12] that could catalyze protein tyrosine oxidation and produce high levels of dityrosine, 3-chlorotyrosine, and 3-nitrotyrosine [13]. Levels of MPO were correlated with poor clinical conditions and sputum production [14], thus demonstrating that extracellular MPO could contribute to hypersecretion, bronchial injury, and respiratory failure in CF. MPO could be a critical parameter in the modulation of inflammation and/or infection because MPO is involved in both microbicidal and proinflammatory activities of neutrophils.
When we have studied purified circulating neutrophils in CF, we provided evidence of a significant increase in MPOdependent oxidant formation in both CF children and their parents as compared to controls, whereas NADPH activity was similar to that of controls. This increased intracellular MPO activity observed in CF homozygotes and heterozygotes was observed at the basal level as well as following PMA or opsonized zymosan stimulation [15]. The fact that this increase was observed in the parents of CF children, which are heterozygous for CFTR mutation but are free of clinical symptoms, led us to the conclusion that the disturbance in MPO activity observed in CF was constitutive, independent from infection. These prior studies demonstrated that increased MPO activity could potentiate inflammation in CF patients.
In the present study, we asked whether a functional MPO promoter polymorphism influenced their clinical outcome. The −463G/A polymorphism is linked to the differences in MPO expression levels [16,17]: the −463G/A promoter polymorphism significantly affects the level of MPO gene expression in bone marrow precursors and in descendant leukocytes. The G allele is more expressing than the A allele. This polymorphism is linked to incidence or severity of inflammatory diseases including atherosclerosis [18,19], Alzheimer's [20][21][22], MPO-ANCA vasculitis [23], HCVinduced fibrosis [23], multiple sclerosis [24], periodontal disease [25], lung cancer [26,27], and myeloid leukemia [24]. This polymorphism is in an Alu element, within a cluster of four hexamer half sites recognized by various nuclear receptors [17] and termed an Alu receptor response element (AluRRE). These hexamers are organized as direct repeats with spacing of two-four-two base pair. The −463A is in hexamer 1 within an estrogen receptor binding site [16], while −463G promotes binding by SP1 transcription factor [17]. The hexamer 3/4 pair is recognized by retinoic acid receptor and PPARγ as heterodimers with retinoid X receptor (RAR-RXR), while the middle DR-4 pair (hexamers 2/3) is recognized by thyroid hormone receptor (TR-RXR). The −463GG genotype has been linked to higher MPO mRNA and protein expression than GA/AA genotypes in primary myeloid leukemia cells and monocyte-macrophages [28], while in transfection assays, the −463G promoter element supported higher expression of a reporter gene than −463A [17]. In a recent study performed in human monocytederived macrophages, we have provided evidence that MPO gene expression was downregulated as iNOS (inductible nitric oxide synthase) was upregulated, thus suggesting a potential connection between these two enzymes [29]. Because NO has physiological importance in airways, a difference in MPO expression levels could be relevant in the pulmonary disease in CF.
MPO could be considered a candidate for potential genetic modifiers of disease severity in patients with CF not necessarily related to infection. Indeed, MPO can also play a significant proinflammatory role in the absence of infection, in atherogenesis, or in neurodegenerative diseases. The mechanisms leading to neutrophil-dominated inflammation in CF patients as well as the relationships with CFTR mutations remain largely unknown. The course of lung disease in patients with CF varies considerably, even among patients with the same CFTR who are receiving standardized care [7]. This variation is largely unexplained. So far, several genes implicated in innate immunity and in the control of inflammation have been studied as potential modifiers of CF [30]. Among them, mannose-binding lectin [31], alphaantitrypsin [32], glutathione S-transferase, TNF-alpha, IL-1 beta, and IL-1 RA have already been studied [33]. Interestingly, a possible link between the NOS1 gene locus and the rate of decline in lung function in CF patients has been described [34]. So far, there is no data available on the influence of MPO genotype on the clinical course of CF.
In the present study, we investigate the influence of the MPO promoter polymorphism on the clinical severity of CF disease. Our results clearly demonstrate that MPO genotype influences the severity of the lung disease in CF patients in the absence of infection, with the GG genotype associated with more severe CF disease. In infected CF patients, the GG genotype was associated with an increase in the circulating neutrophil inflammatory state.

Study population
Eighty eight patients with CF including 45 males and 43 females (mean age: 17.8 ± 0.74 years) were recruited for the study. Informed consent was obtained from patients or their parents and the clinical research was conducted according to the guidelines of the French Institut National de la Santé et de la Recherche Scientifique (INSERM) and the French ethics committee. CF was diagnosed according to the standard criteria including a sweat chloride test. Patient's clinical status was evaluated in CF patients by the Shwachman-Kulczycki scoring system [35] which includes nutritional criteria, the highest value being 100. The pulmonary function was assessed by spirometry and expressed as percent-predicted FEV1 (forced expiratory volume in one second) and FVC (forced vital capacity) normalized for age and height [36]. The severity of lung disease was evaluated according to the chest radiograph and the number of radiologic lesions (obstructive lesions and segmental or diffuse bronchiectasis were counted).
The lung infection was determined in 77 CF patients on the basis of bacteriological analysis of sputum, which was collected according to the methodology previously described [36]. On the basis of sputum bacterial qualitative and quantitative analysis, bronchial infection was defined as bacterial counts ≥ 10 8 colony forming units (CFU)/l in at least 3 cultures at one-month interval, and CF patients were stratified into three groups.
A first group designated "uninfected CF children" included 32 children without history or evidence of P aeruginosa infection. At the time of testing, sputum cultures were all negative and all children were stable and clinically well.
The second group designated "infected CF children" included 45 CF children either infected with Staphylococcus aureus (S aureus) (n = 8) or chronically colonized with P aeruginosa (n = 37).

Analysis of CFTR mutations by PCR and direct sequencing
Genotyping of CF patients was performed as previously described [37][38][39]. In the CF population tested, 73% carried the delta F508 mutation with 50% being delta F508 homozygote, 23% being delta F508 heterozygote with an unknown mutation, and 27% having mutations other than delta F508 at both alleles as previously described [36].

Determination of MPO genotype by allelic discrimination assay
DNA was isolated from leukocytes by proteinase K digestion followed by phenol extraction and ethanol precipitation. DNA (100 ng) was used in the ABI 7900 allelic discrimination assay using dual fluorophore probes which discriminate between alleles based on the single base mismatch. Primers and probes, designed using the Primer Express 2.0 software (ABI), are as follows: forward primer, 5 -AATCTTGGGCTGGTAGTGCTAAA-3 ; reverse primer, 5 -GCCAGGCTGGTCTTGAACTC-3 ; −463A specific probe, 5 -FAM TCCACCTGCCTCAG MGB; and −463G specific probe, 5 -VIC TCCACCCGCCTCA MGB. Probes are labeled at the 5 end with the fluorophores FAM or VIC and are stabilized by a minor groove binding moiety (MGB). Endpoint allelic specific fluorescence was measured on the ABI prism 7900 using the Sequence Detection Systems 2.0 software for allelic discrimination.

Statistical analysis
Statistical analysis was performed using the Statview software. Comparisons were made by analysis of variance (ANOVA) or unpaired Student t test. Data are expressed as mean ± standard error of the mean (SEM).

Frequencies of MPO genotype in CF patients as compared to control population
We determine the MPO genotype for 79 CF children. As shown in Table 1, there is no difference in the percentages of the GG, GA, or AA genotypes in the CF population as compared to the frequencies within normal European populations [16,26,40]. When the cases were stratified by gender, there was no statistically significant difference.

Influence of MPO genotype on clinical parameters of CF disease
Since clinical conditions in CF patients are greatly influenced by recurrent infections, it was relevant to evaluate the influence of MPO genotype within a homogeneous group of CF  Table 2, the group of infected CF children clearly differed from the groups of noninfected CF children on the basis of respiratory scores (FEV or FVC) or infectious score measured as the number of antibiotherapies during the past year. Although not statistically significant, there was a clear difference in the Shwachman and in the radiographic scores between noninfected and infected CF patients; the noninfected CF patients having more severe Shwachman and radio scores. Of note, the mean age of the noninfected CF patients was significantly lower than the age in the infected CF patients. This could be expected since infection occurs in the course of the disease and thus affects older patients. Within each group, either the noninfected or the infected CF patients, we evaluated the influence of MPO genotype. As shown in Figure 1(a), in the group of noninfected CF patients, there is significantly lower FEV in CF patients with GG than GA genotype (73.5 ± 5.6 versus 95.7 ± 8.2, P = .03). Likewise, FVC was significantly lower in CF patients with GG genotype than with GA (80.8 ± 4.0 versus 99.7 ± 6.7, P = .01, resp). As expected, there was more lesions seen on chest radiography in GG than in GA (23.6±8.6 versus 5.5±1.5, resp). Shwachman scores were decreased in the group of GG genotype as compared to GA (64.3 ± 9.1 versus 87.8 ± 3.4, resp). The number of antibiotherapies was higher in GG than in GA (3.5 ± 1.2 versus 2.4 ± 0.8, resp). Of note, the mean age of the CF patients was not different between the groups of GG and GA providing evidence that there was no artifact of older patients in GG group explaining their more severe clinical expression (Table 3).
In contrast, in the group of infected CF patients, there was no significant difference in clinical parameters between GG and GA genotypes evaluated either by respiratory scores (FEV or FVC) (Figure 1(b)) or by radiography scores. There is no difference in FEV in CF patients with GG or GA Taking together, our data strongly suggest that the MPO genotype influences the clinical course of the disease with less severity for the GA allele versus the GG allele in CF patients, in the absence of infection. In contrast, as far as there is an infection and especially a chronic P aeruginosa infection, MPO genotype is not as discriminative, and the GA genotype is not associated with better clinical status.

Influence of gender
Since a number of studies have shown that the influence of MPO genotype could be gender-specific [20,23], we compared the influence of MPO genotype in CF females and CF males. No influence of gender was observed. We confirmed that, in the absence of infection, in either CF females or CF males, the GG genotype was associated with a more severe lung disease and worse clinical conditions. In the group of infected CF patients, there was no significant influence of MPO genotype in CF females or males, thus corroborating the results found in the mixed population.

Influence of the CFTR mutation
CF patients were stratified into two groups according to their CFTR mutation, either homozygotes ΔF508/DF508 or heterozygotes ΔF508/others. The group of patients having the CFTR genotype corresponding to other/other was so small that we could not perform analysis. Within the group of ΔF508/DF508 or within the group of ΔF508/others, we confirmed what we have found for the whole population, which is that the GA phenotype was associated with better clinical parameters as compared with GG, in the absence of infection. In infected CF patients, there was no effect of MPO genotype whatever the CFTR genotype. These results concerning the influence of CFTR mutation on the clinical severity of the disease are not surprising since the CFTR genotype is not related to the inflammatory status of the patient, and it is now well documented that there is no correlation between CFTR genotype and the evolution of lung disease.

DISCUSSION
Our present findings establish for the first time that the GG MPO genotype is associated with more severe clinical expression of CF disease in the absence of infection. The G allele is the higher expressing allele, and the GG genotype is most often associated with worse disease. Thus, the findings in this paper linking GG genotype to worse outcome in CF is consistent with most prior studies.
In CF patients, the association of the higher expressing GG genotype with more severe disease in the absence of infection implies that MPO enzyme contributes to the inflammatory status in noninfected patients. Our data suggest a significant proinflammatory role for MPO in the early phase of CF, in the absence of infection. Several hypotheses can be made to explain the influence of MPO in airways in the absence of infection and the potential mechanisms resulting in the presence of MPO within airways. Firstly, noninfected CF patients means that these patients are non-chronically infected by P aeruginosa or S aureus and they do not have any detectable infection demonstrated by negative bacterial analysis of the sputum at the time of the clinical analysis. In our study, we differentiated between noninfected CF patients and chronically infected patients because their clinical status were significantly different. However, our classification does not rule out some subclinical infection, which could explain the presence of some neutrophils within the lung. Secondly, it has been described that there is a "constitutive" inflammatory process in CF epithelial cells with a basal activation of the transcription factor NF-kB in the absence of infection [41]. It has been reported that epithelial cells from CF patients have the ability to secrete more IL-8, which is a potent chemoattractant for neutrophils [42,43]. It can be hypothesized that inflammatory cytokines could attract neutrophils and/or upregulate MPO in macrophage in the absence of infection. The inhibitory role of MPO on the regulation of NOS might also explain the better clinical conditions associated with the GA genotype (producing less MPO). Indeed, epithelium-derived NO reduced P aeruginosa adherence and enfance killing of bacteria [44], and NOS expression decreased in CF as airway inflammation increases [45]. A combined study of NOS and MPO gene expression and polymorphisms might be relevant to CF and would help to understand the variability of the clinical expression of the pulmonary disease.
In chronically infected CF patients, the GA genotype is not as beneficial as it is in noninfected patients. The observation that GG genotype is not associated with greater resistance to P aeruginosa or fewer infection per year suggests that MPO does not play a critical role in the antimicrobial systems of CF neutrophils. In this situation, many neutrophils are recruited within airways and these fail to kill the bacteria (S aureus or P aeruginosa). One would expect that CF patients having more MPO (GG genotype) would be more efficient in killing bacteria and thus would have less severe pulmonary disease. We do not see any difference in the severity of the pulmonary disease between GG and GA genotypes who are chronically infected.
Our study thus emphasized that MPO contributes significantly to the proinflammatory effects of PMN. In the absence of infection, genotype GG may be more deleterious due to exacerbation of the inflammatory process and production of more oxidants which damage the airways and epithelium. When chronic colonization with P aeruginosa occurs, the increased production of chlorinated oxidants could be an advantage, counterbalancing the negative impact of oxidative damage to the epithelium, such that GG genotype may have both positive and negative impact; thus the difference between GG and GA is less noticeable. Moreover, additional physiopathological mechanisms are involved in the destruction of airways depending on the bacteria, thus masking the clear-cut influence of MPO genotype on the clinical expression of CF disease that we have observed in noninfected patients. Another interesting aspect of our results is the fact that MPO activity could result in the formation of inflammatory mediators by oxidizing mucus components, as it has previously been reported for plasma components in hemodialyzed patients [46]. Indeed, it has been reported that neutrophils components, especially elastase, can stimulate mucus secretion in airways [47] via an oxidant-dependent mechanisms. It would be pertinent to study whether MPOmodified mucins could have some inflammatory activities. This could fit with the data that MPO levels in neutrophils were correlated with sputum production [14], thus suggesting that MPO could be linked with CF mucus hypersecretion.
Several studies on the mechanisms of inflammation in CF have now shown that a genetic disease like CF is not as simple as originally envisioned. The cloning of the CFTR was a major milestone in understanding the molecular basis of CF, but dissecting the pathogenesis of CF has proven far more complicated. The challenge remains to select appropriate gene polymorphisms and investigate their effects in order to achieve a more complete understanding of the pathogenesis of a disease as complex as CF. The identification of a collection of modifying genotypes could lead to a more refined understanding of specific disease. In a study focused on potential genetic modifiers of disease severity in chronic granulomatous disease (CGD) patients, genotype of MPO and Fc gamma RIII were found to be strongly associated with an increased risk for gastrointestinal complications. There were, however, no data on MPO activity in PMN from CGD patients or addressing the possibility that MPO genotype could modulate levels of MPO activity [48].
Understanding the role of modifying genes has considerable potential for determining risk profile that might impact therapeutic decisions. By subclassifying disease, according Table 3: Influence of MPO genotype on clinical parameters of CF disease. The −463G/A MPO polymorphism was determined in 58 CF patients as described in the "methods." The influence of the polymorphism of MPO promoter was studied either in (a) noninfected or in (b) chronically infected CF children. The data are the mean ± SEM. The radio score indicates the number of lesions seen on the chest radiography. The ATB means the number of antibiotherapy treatment during the preceding year. NS: P > .05. 18.6 ± 0.9 6 4 .3 ± 9.1 2 3 .6 ± 8.6 3 .5 ± 1.2 GA (n = 7) 18.0 ± 1. In summary, our hypothesis is that, in CF, the MPO promoter polymorphism −463G/A, which influences the amount of neutrophil MPO, could modulate inflammatory and/or infection processes, thereby modulating the severity of the pulmonary disease. MPO-HOCl is known to oxidize a variety of biological molecules, and can have significant impact at inflammatory sites, independent of infection. This is likely to explain the observed difference between the MPO genotypes in severity of pulmonary disease in noninfected CF patients.


s2fieldsofstudy: Biology Medicine

text:
Specific Remodeling of Splenic Architecture by Cytomegalovirus

Efficient immune defenses are facilitated by the organized microarchitecture of lymphoid organs, and this organization is regulated by the compartmentalized expression of lymphoid tissue chemokines. Mouse cytomegalovirus (MCMV) infection induces significant remodeling of splenic microarchitecture, including loss of marginal zone macrophage populations and dissolution of T and B cell compartmentalization. MCMV preferentially infected the splenic stroma, targeting endothelial cells (EC) as revealed using MCMV-expressing green fluorescent protein. MCMV infection caused a specific, but transient transcriptional suppression of secondary lymphoid chemokine (CCL21). The loss of CCL21 was associated with the failure of T lymphocytes to locate within the T cell zone, although trafficking to the spleen was unaltered. Expression of CCL21 in lymphotoxin (LT)-α–deficient mice is dramatically reduced, however MCMV infection further reduced CCL21 levels, suggesting that viral modulation of CCL21 was independent of LTα signaling. Activation of LTβ-receptor signaling with an agonistic antibody partially restored CCL21 mRNA expression and redirected transferred T cells to the splenic T cell zone in MCMV-infected mice. These results indicate that virus-induced alterations in lymphoid tissues can occur through an LT-independent modulation of chemokine transcription, and targeting of the LT cytokine system can counteract lymphoid tissue remodeling by MCMV.

Introduction
The microarchitecture of secondary lymphoid organs, like the spleen, facilitates effective communication between antigen-presenting cells and T lymphocytes to mount protective immunity to pathogens. The white pulp of the spleen is organized around a central arteriole and is demarcated from the surrounding red pulp by the marginal sinus defining the marginal zone (MZ), wherein antigencapturing macrophages, B cells, and dendritic cells (DC) reside [1]. Two distinct macrophage populations that are present in the MZ include marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM), and these cells localize to the outer and inner border of the MZ, respectively. Within the white pulp, T and B lymphocytes are also segregated into discrete compartments, with T cells comprising the periarteriolar lymphoid sheath (PALS) surrounded by the B cell follicles.
The localization and subsequent organization of T and B lymphocytes to the white pulp is regulated through their interactions with unique populations of chemokine-producing stromal cells [2]. Naive T lymphocytes express high levels of the chemokine receptor CCR7, and upon entering the spleen they migrate toward, and congregate upon, stromal cells that produce the secondary lymphoid tissue chemokines CCL21 (SLC/6Ckine/Exodus-2/TCA4;) and CCL19 (ELC/MIP-3b) [3,4]. Two distinct isoforms of CCL21, encoded by separate gene products that differ by the presence of a serine or leucine at amino acid residue 65, are expressed in C57BL/6 mice. CCL21-ser(a) appears to control the migration/localization of T cells and DC in lymphoid organs [5,6]. Importantly, CCL21-ser regulation of cellular trafficking seems to be due to its high expression levels compared with CCL21-leu(b) in lymphoid tissues, and not a functional difference between these two isoforms in mediating cellular chemotaxis [7]. In contrast, all mature B cells express the chemokine receptor CXCR5, and migrate in response to the production of B lymphoid chemoattractant CXCL13 by radio-resistant stromal cells present in the B cell follicle [8,9].
The development and maintenance of chemokine-expressing stromal cells requires signaling by members of the TNF family of cytokines, lymphotoxin (LT)a (TNFSF1), LTb (TNFSF3), and TNF (TNFSF2) [10], most likely through their activation of transcription factors in the NFjB family [11]. The role of LTab-LTb receptor (LTbR, TNFRSF3) signaling in regulating lymphoid tissue chemokine expression at the developmental, neonatal and adult stage in mice is complex and is still being dissected [2]. Nevertheless, triggering LTbR signaling can induce expression of CCL21 in the spleen of adult mice via activation of the ''noncanonical,'' IKKa/NIKdependent NFjB pathway [12]. Interestingly, LTab signaling has been reported to be critical for the specific induction of CCL21-ser in a mouse model of lung inflammation [13]. However, CCL21-ser induction and the subsequent formation of tertiary lymphoid structures occurs in the lungs of LTadeficient mice infected with influenza, indicating LTab regulation of CCL21-ser is not absolute [14].
Herpesviruses, such as cytomegalovirus (CMV), are effectively controlled in the immunocompetent host, with infection resulting in little overt pathogenicity. However, despite effective host immune defenses, herpesviruses establish a lifelong, persistent infection. Studies of mouse CMV have provided an insightful model, revealing the importance of innate defenses in the control of acute infection, particularly type 1 interferons and specific NK cell subsets [15][16][17], and adaptive immunity mediated by T lymphocytes required for viral clearance and control of latency [18]. Recently, we uncovered a role for the LTab cytokine system in regulating the early type 1 IFN response during MCMV infection, and identified this ''cytokine axis'' as essential for survival of T and B cells [19,20].
These results suggested that CMV might alter the pathways controlling the organization of lymphoid tissue as a strategy to impede efficient trafficking and intercellular communication between responding host cells. Here we show that MCMV disrupts the compartmentalization of lymphocytes and alters trafficking of adoptively transferred T cells by inducing the specific downregulation of CCL21 mRNA in splenic stromal cells. T cell trafficking to the splenic T cell zone is partially restored by activating signaling via the LTbR, indicating that MCMV targets chemokine-controlled architecture as part of its manipulation of host defenses.

Results
To examine whether MCMV infection might modulate lymphoid-tissue organization, splenic architecture was examined by immunohistochemistry during acute viral infection. Infection of C57BL/6 (B6) mice with MCMV induced striking changes in the MZ and white pulp areas by 3 d post-infection ( Figure 1A). MCMV caused a complete loss of MZM and a reduction in the MMM, identified by the ER-TR9 and MOMA-1 markers, respectively. Indicative of virus-induced inflammation, granulocytes (Gr-1/Ly6G hi and CD11b hi ) accumulated in the splenic MZ and red pulp within 48-72 h. Evidence that stromal cell populations in the spleen were altered during MCMV infection was also seen. T cell zone stromal cells can be identified by the expression of gp38 (T1a/podoplanin) [21], a cell surface mucin required for the appropriate development of the lymphatic endothelium [22]. After MCMV infection, an abnormally broad pattern of gp38 staining was observed throughout the white pulp, which in uninfected mice is restricted to the PALS ( Figure 1A). By contrast, the BP-3 (BST-1/CD157; NP_033893) marker identifying stromal cells located mainly in the B cell follicle remained unchanged. Perhaps most conspicuously, B and T lymphocytes were no longer compartmentalized into distinct regions in the white pulp. The effects on microarchitecture progressed in severity as the virus inoculum was increased (unpublished data), with changes evident by 48 h and maximal at 72-96 h, concurrent with the peak of MCMV replication in the spleen of B6 mice [23]. Moreover, these changes did not occur when inactivated (UV-irradiated) virus was used to infect the mice (unpublished data), indicating that either viral gene expression or productive replication is required for modulation of splenic architecture. B6 mice are relatively resistant to MCMV infection, primarily through the action of the cmv1 dominant-acting resistance gene encoding the Ly49H-activating receptor expressed on a subset of NK cells, which help to control viral replication in the spleen [17,24,25]. Importantly, similar modulation of splenic architecture was also seen in Balb/c mice infected with MCMV, indicating that Ly49Hþ NK cells were not required for the observed alterations ( Figure  1B).
The specific changes observed in B and T lymphocyte segregation and the alteration of stromal cells populations in the spleen suggested that expression of the lymphoid tissue organizing chemokines might be altered upon infection with MCMV. Indeed, CCL21 mRNA was specifically decreased in the spleens from B6 mice infected with MCMV ( Figure 2A) with a corresponding loss of CCL21 protein staining in the T cell zone (Figure 2A and 2B). Although stromal cells in the T cell zone coexpress CCL19 and CCL21, the levels of CCL19, and CXCL13 (expressed by stromal cells in the B-cell zone), were largely unaffected after MCMV infection ( Figure 2A). In contrast, expression of several inflammatory chemokines (CCL3, CCL4, and CXCL2) were all highly elevated ( Figure  2A), consistent with the observed recruitment of granulocytes into the spleen [26]. Interestingly, the decreased CCL21expression after MCMV infection was due to a specific effect on the serine isoform of CCL21 (CCL21-ser) ( Figure 2C). The decrease in CCL21-ser was also viral replication dependent, as infection with UV-inactivated MCMV did not result in modulation of CCL21 mRNA levels ( Figure 2A). However, CCL21 mRNA recovered to levels near that of uninfected mice by day 7 post-infection, a time point when virus production has been largely controlled in the spleen ( Figure  2D). These results indicate that MCMV infection results in a specific, but transient, inhibition of the CCL21 chemokine system.
In the spleen, stromal cells in the T-cell zone appear to be the primary producers of CCL21 [2,9]. Stromal cells in the T cell zone of uninfected mice coexpress these two proteins [27]. Interestingly, gp38 mRNA was strongly upregulated following MCMV infection (8-fold to 20-fold) (Figure 2A). These results suggest that MCMV infection does not compromise the viability of CCL21/gp38 coexpressing stromal cells, but instead selectively alters CCL21-ser tran-

Synopsis
The architecture of the spleen and lymph nodes is organized into discrete compartments that promote the immune system's ability to capture pathogens and mount protective defenses. The white blood cells that fill these compartments are positioned in discrete regions by chemoattractants produced by the cells that form the architecture of the spleen (stroma). The white blood cells must be positioned to interact with each other to mount effective host defense. Benedict and colleagues demonstrate that cytomegalovirus, a b herpesvirus, can remodel the architecture of the spleen by specifically inhibiting a key chemoattractant produced by the stromal cells, the chemokine CCL21. This action by the virus impedes efficient contact between cells by altering the movement of T lymphocytes to their correct position within the spleen. However, as a counter strategy, activating the lymphotoxin-b receptor pathway, a cytokine known to control the embryonic development of lymphoid organs, partially restored CCL21 and the ability of T lymphocytes to find their correct position. Thus, infection with this herpesvirus selectively remodels the structure of lymphoid tissues perhaps aiding in the formation of a portal though host defenses.
scription. Alternatively, since gp38 expression was no longer restricted to the PALS, and an intact T cell zone was also absent, it is possible that the increase in gp38 expression occurred on other cell types after MCMV infection.
MCMV dependent modulation of gp38 and CCL21-ser transcription in total spleen RNA indicated that infection could alter gene expression in the stroma. To identify virusinfected cells, a recombinant MCMV that expressed green fluorescent protein (MCMV-GFP) [28] was utilized. This recombinant virus showed an equivalent capacity to induce loss of CCL21 as the Smith strain of MCMV (unpublished data). Flow cytometry revealed that the distribution of cells displaying GFP fluorescence in the hematopoietic compartment at 48 h and 72h post-infection were granulocytes (;18% and 14% at 48 h and 72 h, respectively), macrophages (;10% and 6%), NK cells (;3% and 11%), and DC (;6% and 3%), but very few T or B lymphocytes (, 2% of each) ( Table  1). To accurately determine these percentages, it was necessary to compare the fluorescence of individual hematopoietic cell subsets in the FL-1 channel from mice infected with MCMV-GFP to cells from mice infected with MCMV-Smith due to changes in the autofluorescence of different cellular subsets following virus infection (see Figure S1, specifically Figure S1C). In addition, the expression of the macrophage marker F4/80 was downregulated in mice infected with MCMV, as has been reported previously [29], and this was accounted for when assessing the percentage of infected macrophages (see Figure S1A and S1B).
To quantify the relative tropism of MCMV for the splenic hematopoietic and stromal cells, the spleen was separated into two fractions by extrusion through a nylon filter. An ;500-fold enrichment of mRNA for CCL21 and CXCL13, and ;200-fold for CCL19 was observed in the fraction retained by the filter (stroma) when compared to hematopoietic cells in the extruded fraction from uninfected mice ( Figure 3A). MCMV infection altered CCL21-ser expression (28-fold decrease) in the stromal fraction, with no reduction in CCL19 or CXCL13 expression. Viral gene expression as determined by analysis of IE1 (immediate early gene-1) largely fractionated with stromal cells (;9-fold abundance over the level of IE1 in the hematopoietic compartment) (Figure 3 A). Together, these data indicate that MCMV preferentially infects the splenic stromal compartment, and suppressed CCL21-ser. To further map the location of MCMV-infected cells in the spleen, a detailed immunohistochemical analysis was performed ( Figure 3B). The majority of cells expressing high levels of GFP were localized in the red pulp and MZ region of the spleen. Many GFP þ cells in the red pulp costained for platelet-endothelial-cell adhesion molecule-1 (PECAM-1), but not for a marker of the reticular fibroblastic network (ER-TR7), suggesting that the virus infected EC. High magnification analysis in the MZ revealed that some GFP-expressing cells also co-expressed mucosal addressin cell adhesion molecule (MAdCAM), while other cells displayed morphology typical of GR-1þ granulocytes previously localized to this region (see Figure 1). Lowmagnification analysis revealed a sparse distribution of GFP-expressing cells in the splenic white pulp compared with the MZ and red pulp. When the white pulp area was examined at higher magnification, the GFP þ -MCMV-infected cells were morphologically hematopoietic cells without typical stromal cell characteristics. Although gp38 levels were upregulated in the white pulp of virus-infected mice (see Figure 1), a small fraction of cells coexpressing gp38 and GFP were sporadically observed ( Figure 3B). These data indicate that MCMV preferentially infects splenic endothelium and hematopoietic cells localized in the red pulp and MZ.
LTab-LTbR signaling is required for the development of CCL21 and gp38-expressing stromal cells in the spleen [30], which prompted us to examine the effect of MCMV infection on CCL21 and gp38 expression in the spleens of LTa-   deficient mice ( Figure 4A). Although LTa-deficient mice have significantly lower basal levels of CCL21 expression when compared with wild-type mice (;20-fold reduced), MCMV infection resulted in a further decrease (20-fold), occurring over a similar timecourse to that seen in control mice. Similar to wild-type mice, the CCL21ser isoform was specifically affected by MCMV infection (unpublished data). By contrast, the basal transcription level of gp38 was modestly reduced in spleens of uninfected LTa-deficient mice compared with wild-type mice (1.8-fold reduced compared with wild-type), yet 3 d after MCMV infection gp38 was dramatically upregulated in both (16-fold in wild-type; 84-fold in LTaÀ/ À). Additionally, the expression of LTb in total spleen mRNA was determined 3 d post-infection in wild-type mice infected with various amounts of MCMV ( Figure 4B). At lower doses of virus (2 3 10 4 pfu), CCL21-ser expression was significantly reduced (;5-fold), but expression of LTb was minimally affected (;20% reduction). With increasing inoculum of MCMV, CCL21-ser levels further decreased, as did LTb expression. However, at the two higher doses of MCMV the reduction in LTb was proportional to the loss of total splenocytes (hematopoietic cells) ( Figure 4B), suggesting that the reduction in LTb mRNA probably reflected a loss of cells. This loss of lymphocytes also contributes to the alteration in the relative percentages of hematopoietic cell subsets observed in the spleen after MCMV infection (see Table 1). This lymphocyte ''death'' may be linked to LTab-LTbR signaling required for sufficient induction of IFNb, which contributes to lymphocyte survival [20]. These results suggest that the MCMV-induced effects on CCL21 and gp38 expression can occur largely in the absence of an intact LTab cytokine system, and that reduced LTab expression is likely not responsible for the decrease in CCL21-ser. It is not clear whether the main effector chemokine that controls T cell localization to the PALS is CCL19 or CCL21. The specific suppression of CCL21-ser expression, but not CCL19 expression, after MCMV infection, presented an opportunity to distinguish the function of these two chemokines. MCMV infection should impair the homing of T cells to the PALS if CCL21 is the critical chemokine regulating this trafficking. To assess T cell trafficking in vivo, flow cytometry and immunohistochemistry were used to monitor the movement of adoptively transferred T cells labeled with a fluorescent dye (CFSE) ( Figure 5). Naive T cells transferred into mock-infected mice showed rapid localization (within 2 h) to the PALS, while T lymphocytes transferred into MCMVinfected mice were unable to home to the T cell zone ( Figure  5B), although equivalent numbers of labeled T cells from either mock-or MCMV-infected mice migrated to the spleen ( Figure 5A). Importantly, T cells isolated from MCMVinfected mice were fully capable of localizing to the PALS when injected into uninfected mice, indicating that the function of the CCL21 receptor (CCR7) was unaffected in splenic T cells after MCMV infection (unpublished data). However, T cells transferred to MCMV-infected mice were largely restricted to the MZ and red pulp ( Figure 5B), similar to the trafficking patterns of T cells in paucity-of-lymphnode-T cells (plt) mice (which lack expression of CCL21-ser and CCL19) and CCR7-deficient mice [3,4].
The observation that MCMV modulation of CCL21 and gp38 transcription occurred in both wild-type and LTadeficient mice suggested that the LTab-CCL21 signaling ''axis'' might still be intact in the lymphoid tissue of these infected mice. To test whether this pathway was active, we used an agonistic antibody that activates LTbR signaling [12,20] to induce CCL21 expression. The anti-LTbR antibody was co-injected at the time of MCMV infection. CFSE-labeled T cells were adoptively transferred 3 d post-infection, and spleens were harvested for analysis of T cell localization and CCL21 expression. A single injection of anti-LTbR resulted in a modest restoration of CCL21 mRNA levels (;2-fold), without any change in CCL19 or CXCL13, which was restricted to the CCL21-ser isoform ( Figure 6A). A commensurate increase in the localization of transferred T cells to the white pulp also occurred in anti-LTbR-treated mice (Figure

6B). This result indicates that the LTbR pathway remains operable during MCMV infection. Activating the LTbR pathway was sufficient to direct trafficking of T cells to the PALS counteracting viral-induced alterations in lymphoid tissues. The induction of CCL21 by the LTbR agonist suggests
that the MCMV-induced decrease in CCL21-ser expression is responsible for the inability of transferred T cells to localize to the PALS.

Discussion
The cytokine pathways required for the development and maintenance of organized lymphoid tissue also contribute to efficient host defense against viral pathogens. Our previous observations that MCMV exhibited increased virulence in mice lacking an intact LTab-cytokine system [19,20], a system critical for the development and maintenance of lymphoid tissue architecture, prompted us to examine whether viral infection might alter the structure of lymphoid organs in wild type mice. MCMV infection induced several prominent changes in the microarchitecture of the spleen, including alterations in MZ macrophage populations, a complete loss of B and T cell segregation in the white pulp commensurate with a specific and dramatic loss in CCL21-ser expression, and increased expression of the PALS stromal cell marker gp38. We identified the PECAM-1-and MAdCAM-expressing EC as major cellular targets of the splenic stroma infected with MCMV. The EC expressing these markers in the MZ contribute to the ''threshold'' that T cells must cross to accumulate in the PALS after entry into the spleen through the MZ sinus. Regulation of T cells trafficking is accomplished in part through the expression of key integrins and chemokines, including CCL21 [31].
The phenotype of mice deficient in CCR7 [3] or harboring the mutation plt, which lack CCL21-ser and CCL19 [6,27], provided solid evidence that these chemokines regulate T cell trafficking to the white pulp and maintenance of T and B cell segregation. Additionally, T/B cell zones are disorganized in the spleens of mice deficient in LTab signaling, which also show marked reductions in CCL19 and CCL21-ser expression and CXCL13 [13,32]. Unfortunately, the construction of mice singly deficient in either CCL21-ser or CCL19 signaling has not been achieved. Here we identified that the b-herpesvirus CMV can in a highly specific fashion inactivate CCL21-ser at the transcriptional level. MCMV had no effect on CCL21-leu or CCL19. Infection with MCMV induced a blockade in T cell trafficking to the PALS, implicating that CCL21-ser is the essential player in maintaining T lymphocyte organization in the white pulp. However, we cannot rule out the possibility that MCMV inhibits additional, unidentified factors that regulate T cell localization in the spleen. Interestingly, human CMV has also been reported to modulate CCR7-dependent signaling. Human CMV infection of immature DC cells inhibits their ability to upregulate CCR7 expression, resulting in decreased chemotaxis in response to CCL19/CCL21 [33]. Therefore, both mouse and human CMV appear to specifically target this chemokine system, albeit by distinct mechanisms.
In addition to the dramatic disruption in lymphocyte organization observed after MCMV infection, significant alterations were also seen in MZ architecture. The MZM and MMM populations are one of the first cell types to encounter blood borne antigens/pathogens that enter the spleen via the MZ sinus, and are likely to play an important role in antigen capture and presentation. In addition, these MZ macrophages have been shown to be the major early producers of type I interferon (IFNab) after herpes simplex Right Panel, mice were infected with 1.5 3 10 5 pfu MCMV and expression of the various CCL21 isoforms was determined. CCL21u, universal CCL21 primers that amplify both the leucine (leu) and serine (ser) isoforms; p ¼ 0.02 (B) Shown are representative spleen sections from the same mice that were analyzed in the right panel of (A). Sections were incubated with an antibody to detect MMM (MOMA-1, MMM, blue), and adoptively transferred, CFSE-labeled T cells are green. The number of CFSE labeled T cells located per WP area after transfer was analyzed (n ¼ number of WP regions analyzed per spleen), and differences between groups of infected mice 6 antibody is statistically significant to a p-value , 0.0001. DOI: 10.1371/journal.ppat.0020016.g006 virus infection [34]. MCMV infection disrupts MZ organization within 2-3 d post-infection, and these changes could be due to several mechanisms. One possible explanation for the inability to detect ER-TR9 þ MZM could be that ER-TR9 is downregulated from the cell surface and subsequently degraded during MCMV infection. ER-TR9 has recently been identified as the SIGN-R1 lectin (NP_573501) [35], a homologue of human DC-SIGN. DC-SIGN mediates infection of DC by human CMV through the binding of the viral glycoprotein B [36], and SIGN-R1 is internalized from the surface of MZM after binding to dextran or polysaccharides of Streptococcus pneumonia [37]. However, the loss of ER-TR9 expressing cells might be linked to the specific downregulation of CCL21-ser following MCMV infection, in agreement with the recent observation that plt mice have decreased numbers of MZM [31]. As potential support for this mechanism, we observed a ;9-fold reduction of ER-TR9 mRNA in the spleen 3 days following MCMV infection (data not shown), suggesting that this macrophage population may not be present in the spleen at this time point. The exact reason for the disruption of MOMA-1 þ MMM in the MZ after MCMV infection is unknown at this point, but the broader, more disorganized pattern is not unlike that recently reported in mice lacking a receptor for sphingosine-1phosphate-3 [38].
The dramatic effects on splenic architecture seen during viral infection raise the question of whether these events are specific to MCMV. Lymphoid tissues are dynamic structures that show alterations in both the hematopoietic and stromal compartments during immune responses. Infection of mice with other pathogens such as lymphocytic choriomeningitis virus [39] and Leishmania donovani [40] has been reported to alter lymphoid tissue architecture, but these changes occur at significantly later times after infection (weeks to months) and appear somewhat more ''nonspecific'' due to the loss/death of cell populations in the MZ or stroma. Kataki and colleagues demonstrated that the architecture of the lymph node changes after immunization with inert antigen with a strong adjuvant resulting in alteration of the boundary-separating T and B cells, and changes in the fibroblast reticular network [41]. Thus, for some tissue remodeling the presence of a replicating pathogen is not a prerequisite. Interestingly, changes in ER-TR7 expression were recapitulated to some extent in cultured lymph node fibroblastic reticular cell lines through activation of LTab and TNF signaling pathways [41]. Therefore, the changes in lymphoid tissue architecture during MCMV infection may represent viral exacerbation of ''normal'' host processes regulated by TNF-family cytokines. Nevertheless, it seems likely that MCMV may also employ specific strategies to disrupt lymphoid tissue architecture, which may facilitate replication, dissemination, and establishment of viral persistence. Support for this hypothesis comes from the fact that the disruption in architecture was most dramatic during the peak of acute viral replication, with recovery of lymphoid tissue architecture and CCL21 expression as MCMV replication was controlled.
MCMV-induced alterations in lymphoid tissue architecture were accompanied by specific enhancement of stromal cell expression of gp38. The restricted expression of gp38 to stromal cells located in the PALS of the white pulp has been known for some time [21]. Expression of this mucin in the PALS is defective in mice deficient in LTa [30]. The concurrent reduced levels of CCL21 and CCL19 expression in the spleens of LTa deficient mice is thought to be due to a defect in the maturation of PALS stromal cells, which coexpress these chemokines and gp38 [2,30]. Gp38 analyzed by immunohistochemistry is undetectable in LTa-deficient mice [30], yet only a 2-fold loss of mRNA is observed, raising the possibility that stromal cells outside the T cell zone may express gp38 that is either not detectable by standard immunohistochemical techniques, or the cellular localization of gp38 may be regulated post-transcriptionally by LTab signaling (as was recently shown for the regulation of ER-TR7 expression [41]). The selective advantage of altering gp38 expression by either the host or the pathogen is unknown.
The LTab-LTbR system plays an essential role in the development and maturation of the splenic architecture, but may provide a limited contribution in the adult organ. Pharmacological inhibition of LTab signaling in adult mice only reduces CCL21 expression by 2-fold in the spleen [32]. Moreover, the homeostatic maintenance of splenic CCL21 expression in adult mice can be restored by bone marrow reconstitution with LTa-deficient bone marrow [30]. In contrast, pharmacological inhibition of LTab signaling in neonatal mice dramatically decreases splenic CCL21 expression (5-fold to 20-fold reduction) [30], and LTa or LTb deficient mice are severely reduced (20-fold to 30-fold) in CCL21 expression. Taken together, these results support a model where LTab is critical during the embryonic development and postnatal maturation of CCL21 producing stromal cells, but once this cell population is ''established'' the contribution of LTab in maintaining CCL21 expression diminishes. Administration of an agonistic antibody to LTbR at the time of MCMV infection partially restored expression of CCL21-ser in the spleen and promoted increased localization of adoptively transferred naïve T cells into the PALS. These results indicate that although the LTab-CCL21 signaling ''axis'' may not maintain CCL21-ser expression in the adult spleen, the pathway can be modulated pharmacologically during a virus infection, as demonstrated previously in uninfected mice [12,13]. Importantly, these data also highlight targeting the LTbR as a potential avenue for manipulation of chemokine expression and potential restoration of some aspects of lymphoid tissue structure in the context of viral infection. This approach might prove useful in contexts where congenital or reactivated CMV infection poses significant clinical risks.

Materials and Methods
Mice and virus. C57BL/6 (B6) mice were purchased from the Jackson Laboratories (Bar Harbor, Maine, United States). LTadeficient mice were described previously [42] and were bred at LIAI. Female mice aged 6-8 wk were used. MCMV (Smith strain) and MCMV-GFP (Gift from J. Hamilton) [28] were prepared in salivary glands and infectious virions determined by plaque assay on NIH 3T3 cells as described in [43]. Virus was administered to mice via the intraperitoneal route.
Cryosections were washed in PBS and stained for 60 min with the following uncoupled, biotinylated, or FITC-coupled primary anti- Quantitative PCR. Levels of gene expression were measured by quantitative RT-PCR using a Stratagene Mx 4000 and SYBR green reagent (BioRad). Spleens from mock or MCMV-infected mice were snap frozen in liquid nitrogen before homogenization in Trizol reagent (Invitrogen) and total cellular RNA isolated and quantified before performing DNAse digestion and reverse transcription as described [19]. Primers to specifically detect CCL21ser and CCL21leu were described previously [7]; all other primer sequences are available upon request.
Splenic stromal cell fractionation. Spleens from mock or MCMVinfected mice were removed aseptically at day 3 post-infection. Spleens were perfused with 1ml/spleen of collagenase (100 U/ml) (CLSIII, Worthington Biochemical, Freehold, New Jersey, United States), resuspended in Hanks' Balanced Salt Solution (HBSS, 100U/ ml) (Invitrogen) containing Ca 2þ and Mg 2þ . After perfusion with collagenase, spleens were incubated for 30 min at 37 8C in collagenase (400 U/ml) solution (1 ml/spleen). Spleens were then extruded through a 70-lm nylon filter strainer (BD Falcon, BD Biosciences, Franklin Lakes, New Jersey, United States). After extrusion, the cell strainer was washed with sterile PBS (Invitrogen) followed by the direct lysis of filter-bound ''stromal cells'' with 1 ml of Trizol (Invitrogen). Cells that were extruded through the filter (''hematopoietic cells'') were washed with sterile PBS (Invitrogen,) and then lysed in Trizol (Invitrogen) (10 7 cell/ml). Total cell RNA isolation and subsequent reverse transcription was performed according to the manufacturers' protocols and as described in [19]. The relative ''contamination'' of filter-bound stromal cells with hematopoietic cells was estimated by analysis of genes preferentially expressed in the hematopoietic cells, including CD80, IL-7Ra, LTa and LTb. Using these markers, mRNA expression in the hematopoietic fraction was found to be 4.43, 5.33, 6.33 and 3.53 enriched in each marker, respectively, relative to the filter retained stromal cell fraction. IL-7 and gp38 mRNAs, two genes expressed preferentially in the splenic stroma, were present at 143 and 173 higher levels in the stromal cells, respectively. These data indicated the filter-bound stromal fraction from uninfected mice contains ;15%-30% contaminating hematopoietic cells, and 4%-7% contaminating stromal cells in the hematopoietic fraction based on the assumption that these marker genes are expressed exclusively in their respective compartments.
T cell transfer. Splenic T cells were purified by negative selection using a ''Pan T Cell Kit'' (Miltenyi Biotech, Auburn, California, United States) following the manufacturer's protocol, and were labeled for 10 min at 37 8C with 1 lM CFSE (5-[and 6-] carboxyfluorescein diacetate succinimidyl ester; Molecular Probes). CFSE-labeled T cells (10 7 ) were injected via the lateral tail vein, and spleens were recovered 2 h later for immunohistochemistry and flow cytometric analysis. MCMV-infected mice receiving anti-LTbR agonistic antibody (3C8) were injected once with 100 lg i.p. at the time of infection.
Spleen sections were stained with MOMA-1 and Thy1.2 to delineate the white pulp boundary and confirm the presence of T cells preceding the analysis of CFSE-labeled cell number within the white pulp. Immunofluorescent images were recorded by a digital fluorescence microscopy system (Intelligent Imaging Innovations, Denver, Colorado, United States), including a Zeiss Axiocam Mrm charge-coupled device camera and a Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Thornwood, New York, United States) and emission filter wheels (Sutter Instrument, Novato, California, United States) with narrow band optical filters (Chroma Technology, Rockingham, Vermont, United States). All components were controlled by SlideBook software (Intelligent Imaging Innovations), and the same software was used for image capture and analysis of the number of CFSE(þ) T cells within the white pulp areas. and ''MCMV-macrophage'' (Gr-1 lo F4/80 int CD11c lo ) populations was analyzed in mock and MCMV-infected mice at 48 h post-infection (similar results were also seen at 72 h, unpublished data). The dot plots show that the expression of F4/80 decreased on the cell population presumed to be splenic macrophages in MCMV-infected mice (i.e., ''MCMV-macrophages'') as reported previously [29], while the expression of GR-1 in these cells remained the same. However, the altered expression of CD11b in macrophages from these three experimental groups also raised the possibility that this is a distinct cell population only seen in the spleen of MCMV-infected mice, potentially recruited from the blood or bone marrow. Mock (red), MCMV-Smith (blue), and MCMV-GFP (green). (C) The percentage of GFP-expressing cells was determined in individual cell populations from spleens of MCMV-GFP-expressing mice (a summary of data is presented in Table 1). For this analysis, GFP-expressing cells were defined as cells that displayed higher levels of fluorescence in the FL-1 channel when compared with mice infected with MCMV-Smith virus (not mock-infected mice). The increase in the autofluoresence occurred in DC, granulocyte, and NK cell populations following infection with MCMV-Smith. DC and granulocytes were phenotypically defined as in (A). For analysis of macrophages, FL-1 fluorescence displayed in histograms was analyzed by gating on the macrophages (Gr-1 lo F4/80 hi CD11c lo ) in mock-infected mice and compared with the ''MCMV-macrophages'' (Gr-1 lo F4/80 int CD11c lo ). GFP expression in NK cells (DX5 þ CD3 À ), and lymphocytes (72 h only) was also analyzed. The higher percentage of cells expressing GFP as fraction of the total splenocytes observed at 72 h after infection (9.9%) compared with 48h (1.6%) is considered in the context of the increased lymphocyte death at this time point (shown in Figure 4B). The high level of FL-1 autofluorescence in the DC from mice infected with MCMV-Smith at both 48 h and 72 h post-infection may be due to activation and/or apoptosis of these cells, and a change in the ''tightness'' of this gated population is evident in MCMV-infected mice. Virtually all splenic DC matured (defined by upregulation of CD80, CD86, CD40, and MHC II) upon infection with MCMV at this dose by 16 h post-infection (unpublished data). Granulocytes expressed higher levels of GFP at 72 h compared with 48 h after infection, suggesting that MCMV-GFP replicates in these cells. However, increased phagocytosis of GFP protein or GFP-expressing cells by granulocytes cannot be excluded. Data shown is from splenocytes pooled from two individually infected mice, and is representative of the results seen in three independent experiments. Found at DOI: 10.1371/journal.ppat.0020016.sg001 (1.0 MB PPT).


Fishy Cooperation

It is commonly thought that animals can be arranged along a ladder of intelligence—a sort of modern-day Scala Naturae— with humans inevitably at the top, followed by our close relatives, the primates, all the way down to fi sh and other slimy creatures. Over the past decade, this ladder has been challenged by claims of high intelligence and great social complexity in other animals. For example, spotted hyenas (Crocuta crocuta) establish hierarchies in which dominant females support the rank contests of their daughters. Bottlenose dolphins (Tursiops aduncus) form " political " coalitions every bit as complex as those of chimpanzees. Caledonian crows (Corvus moneduloides) not only use tools in the wild, but also modify tools in the lab, an ability once thought to defi ne humans. And now come the fi sh. It started with a provocative challenge to primate supremacy with the claim that " culture " (that is, socially transmitted behavior) is at least as well developed in fi sh as it is in primates. While this may be a bit of an exaggeration, a new study on cooperative behavior by Redouan Bshary and his colleagues really makes one wonder if there is anything fi sh cannot do. The article describes the astonishing discovery of coordinated hunting between groupers (Plectropomus pessuliferus) and giant moray eels (Gymnothorax javanicus) in the Red Sea. These two species make a perfectly complementary pair. The moray eel can enter crevices in the coral reef, whereas the grouper hunts in open waters around the reef. Prey can escape from the grouper by hiding in a crevice and from the moray eel by leaving the reef, but prey has nowhere to go if hunted by a combination of these two predators. The article offers a description and accompanying videos, such as the one showing a grouper and eel swimming side by side as if they are good friends on a stroll. It also offers quantifi cation, which is truly hard to achieve in the fi eld, of the tendencies involved in this mutually benefi cial arrangement. The investigators were able to demonstrate that the two predators seek each other's company, spending more time together than expected by chance. They also found that groupers actively recruit moray eels through a curious head shake made close to the moray eel's head to which the eel responds by leaving its crevice and joining the grouper. Groupers showed such …

It is commonly thought that animals can be arranged along a ladder of intelligence-a sort of modern-day Scala Naturaewith humans inevitably at the top, followed by our close relatives, the primates, all the way down to fi sh and other slimy creatures.
Over the past decade, this ladder has been challenged by claims of high intelligence and great social complexity in other animals. For example, spotted hyenas (Crocuta crocuta) establish hierarchies in which dominant females support the rank contests of their daughters. Bottlenose dolphins (Tursiops aduncus) form "political" coalitions every bit as complex as those of chimpanzees. Caledonian crows (Corvus moneduloides) not only use tools in the wild, but also modify tools in the lab, an ability once thought to defi ne humans.
And now come the fi sh. It started with a provocative challenge to primate supremacy with the claim that "culture" (that is, socially transmitted behavior) is at least as well developed in fi sh as it is in primates. While this may be a bit of an exaggeration, a new study on cooperative behavior by Redouan Bshary and his colleagues really makes one wonder if there is anything fi sh cannot do.
The article describes the astonishing discovery of coordinated hunting between groupers (Plectropomus pessuliferus) and giant moray eels (Gymnothorax javanicus) in the Red Sea. These two species make a perfectly complementary pair. The moray eel can enter crevices in the coral reef, whereas the grouper hunts in open waters around the reef. Prey can escape from the grouper by hiding in a crevice and from the moray eel by leaving the reef, but prey has nowhere to go if hunted by a combination of these two predators.
The article offers a description and accompanying videos, such as the one showing a grouper and eel swimming side by side as if they are good friends on a stroll. It also offers quantifi cation, which is truly hard to achieve in the fi eld, of the tendencies involved in this mutually benefi cial arrangement. The investigators were able to demonstrate that the two predators seek each other's company, spending more time together than expected by chance. They also found that groupers actively recruit moray eels through a curious head shake made close to the moray eel's head to which the eel responds by leaving its crevice and joining the grouper. Groupers showed such recruitment more often when hungry.
Given that cooperative hunting increases capture success for each of the two predators, and that they don't share with each other but swallow the prey whole, their behavior seems a form of "by-product mutualism," defi ned as a form of cooperation in which both parties achieve rewards without sacrifi cing anything for the other. They are both out for their own gain, which they attain more easily together than alone.
The observed role division comes "naturally" to two predators with different hunting specializations, and is therefore far simpler to achieve than for members of the same species. Also, recruitment is quite common in the animal kingdom-for example, primates have specialized signals to solicit each other's support in fi ghts. What is truly spectacular about this study is that the entire interaction pattern-two actors who seemingly know what they are going to do and how this will benefi t them-is not one we usually associate with fi sh. This is probably because we tend to develop cognitively demanding accounts for our own behavior and believe that absent the same cognition, the behavior simply cannot take place. It is very well possible, however, that our accounts overestimate the amount of intelligence that goes into complex behavior. Moreover, we have a tendency to underestimate the intelligence of animals at lower rungs of the evolutionary ladder.
In fact, it is the ladder idea itself that is wrong. The best way to approach animal intelligence is from an evolutionary and ecological perspective focused on the tasks that each species faces in nature. In this regard, these two reef predators show us that if it comes to survival, highly intelligent solutions are within the reach of animals as different from us as fi sh.