| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1
Divisions of
*
Molecular Immunology and
Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(PKC) as an important intermediate in signaling pathways leading to T cell activation, proliferation, and cytokine production. However, the importance of PKC to many T cell-driven inflammatory responses has not been demonstrated. In this study we show that although PKC is required for the development of a robust lung inflammatory response controlled by Th2 cells, it plays a lesser role in the development of a similar lung inflammatory response controlled by Th1 cells. PKC-deficient mice were strongly compromised in generating Th2 cells and exhibited reduced airway eosinophilia and Th2 cytokine production in lungs. PKC was required for the initial development of Th1 cells, with these cells exhibiting delayed kinetics of differentiation and accumulation. However, with recall Ag challenge via the airways, this defect was overcome, and lung infiltration and Th1 cytokine production were largely unimpaired in PKC-deficient animals. These data suggest that PKC can play roles in aspects of both Th2 and Th1 responses, but lung inflammation induced by Th2 cells is more dependent on this protein kinase than lung inflammation induced by Th1 cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(PKC),4 a Ca2+-independent isoform of the PKC family of intracellular serine/threonine kinases, may be the major signaling moiety linking TCR- and CD28-specific signals necessary for efficient T cell activation (1, 2). PKC is more strongly expressed in T cells than other cell types and is translocated to the site of contact between T cells and APCs, where it colocalizes with the TCR/CD3 complex in the central core of the immunological synapse after TCR/CD28 ligation (3, 4, 5, 6). Furthermore, studies using pharmocological inhibitors and PKC antisense RNA and overexpression studies using dominant negative and constitutively active mutant proteins have established a role for PKC in activation of AP-1 (7) and NF-
B (8, 9). PKC also synergizes with calcineurin to activate NFAT and IL-2 transcription (10, 11), and with Vav to mediate IL-4 gene expression in response to CD28 costimulation (12).
In further support of its important role in T cell activation, peripheral T cells isolated from PKC deficient mice (PKC–/–) demonstrate profound defects in TCR/CD28-induced IL-2 production and proliferation (4, 13). TCR/CD28-induced NF-B and AP-1 activation is significantly reduced in PKC-deficient T cells (4, 13), and NFAT activation is abrogated (13).
Although these in vitro data strongly support the idea of a critical function for PKC in T cell responses, there are few studies to date showing the importance of this molecule in T cell-driven immune responses in vivo. A recent report in PKC knockout mice demonstrated normal generation of lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells and normal Ab responses after vesicular stomatitis virus (VSV) infection, but that PKC-deficient CD8 T cells were impaired in generating CTL activity after peptide immunization (14). This suggests that there may be differential requirements for PKC depending on the subset of T cells responding or the inflammatory conditions that accompany T cell priming. In the present study we used PKC-deficient mice and determined their susceptibility to lung inflammation controlled by either Th2 or Th1 cells. Our results clearly demonstrate that PKC is required for optimal Th2 responses in vivo, including the production of high levels of IL-4 and IL-5, the recruitment of large numbers of eosinophils to the airways, goblet cell hyperplasia, and mucus production. Development of Th1 cells was impaired and delayed in kinetics, but neutrophilia and production of IFN-
in lungs were relatively normal after recall Ag challenge. These data provide clear evidence that this enzyme plays a major role in the development of Th2 cells and allergic asthmatic reactions, but is not absolutely essential for Th1 cells to be generated. This suggests that a requirement for PKC may be overcome in certain situations that involve strong Th1-inducing stimuli.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The studies reported here conform to the principles outlined by the animal Welfare Act and the National Institutes of Health guidelines for the care and use of animals in biomedical research. PKC-deficient mice (PKC–/–; originally on a 129-C57BL/6J background) were a gift from Dr. D. Littman (Howard Hughes Medical Institute, Skirhall Institute of Biomolecular Medicine, New York, NY;4) and were backcrossed for more than five generations onto the C57BL/6 background. These mice have no defect in thymic selection and bear normal numbers of peripheral T cells. Wild-type (wt; PKC+/+) C57BL/6 mice were used as controls.
Induction of airway inflammation
Mice were sensitized and challenged as described previously (15) to induce a Th2 response or with a modification described below to induce a Th1 response. Briefly, mice were either sensitized by i.p. injection of 20 µg of OVA protein (chicken egg albumin; Sigma-Aldrich, St. Louis, MO) adsorbed to 2 mg of aluminum hydroxide (alum; Pierce) in PBS on day 0 for a Th2 response or sensitized by s.c. injection of OVA (50 µg/ml) in CFA at the base of the tail for a Th1 response. Nonsensitized (control) mice received 2 mg of alum in PBS or CFA alone. On day 25 all mice were challenged via the airways with OVA (5 mg/ml in 15 ml of PBS) for 30 min, once a day for 4 consecutive days, by ultrasonic nebulization. Mice were killed 18–24 h after the last aerosol challenge and were assessed for inflammation of the lung.
Measurement of airway hyper-responsiveness (AHR)
AHR was measured in vivo 1–3 h after the last aerosolized OVA exposure by recording respiratory curves by whole-body plethysmography (Buxco Electronics, Wilmington, NC) in response to inhaled methacholine (1.25–10 mg/ml; Aldrich, Milwaukee, WI) as described previously (15).
Bronchoalveolar lavage (BAL) measurements
Inflammation was assessed by BAL. Lungs were lavaged five times with 1-ml aliquots of PBS containing 2% BSA. The recovered BAL fractions were centrifuged; cells were counted, then pelleted onto glass slides by cytocentrifugation. Differential cell counts were performed on Hema 3 (Fisher Scientific, Pittsburgh, PA)-stained cytospins using standard morphology and staining characteristics. BAL was assessed for cytokine content by standard ELISA protocols using commercially available Abs or those produced in-house (15). Standard curves were constructed with purified rIL-4 (PeproTech, Rocky Hill, NJ), IL-5 (eBiosciences, San Diego, CA), IL-13 (R&D Systems, Minneapolis, MN), and IFN- (PeproTech). All data were collected 24 h after the final Ag challenge unless otherwise stated.
Characterization of lung histology and cellular infiltrates
Lung sections were prepared and analyzed as described previously (15). Lungs were removed and postfixed in 10% zinc-buffered formalin (Protocal; Fisher Diagnostics, Middletown, VA) and paraffin-embedded, and sections (5 µm) were stained with H&E for quantitation of inflammatory infiltrates or with periodic acid-Schiff (PAS) for quantitation of bronchial mucus gland hypertrophy. A semiquantitative scoring system was used to grade the size of lung infiltrates, where +5 signified a large (more than three cells deep) widespread infiltrate around the majority of vessels and bronchioles, and +1 signified a small number of inflammatory foci. To ensure comparable analysis between different groups, we analyzed six to eight randomly selected airways per lung section per animal, and an average inflammatory score was obtained for the entire lung tissue.
Assay of T cell function ex vivo
Peribronchial lymph nodes (PBLNs) were collected at the time of lung harvest. Lavaged lungs were digested with HBSS (Invitrogen Life Technologies, Gaithersburg, MD) supplemented with 3 mg/ml collagenase (type V; Roche, Indianapolis, IN), 0.1 mg/ml DNase (Sigma-Aldrich), 100 µg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA), and 100 U/ml penicillin (Invitrogen Life Technologies) for 60 min at 37°C. After lysing RBCs with ACK lysis buffer, PBLN and lung cells were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (Omega Scientific, Tarzana, CA), 1% L-glutamine (Invitrogen Life Technologies), 100 µg/ml streptomycin, 100 U/ml penicillin, and 50 µM 2-ME (Sigma-Aldrich). Lung cells (8 x 105 cells/well), or PBLN cells (2 x 105 cells/well) were plated in round-bottom, 96-well microtiter plates in 200 µl with increasing concentrations of OVA (10–100 µg/ml) for 96 h at 37°C. After 84 h, 1 µCi of [3H]thymidine (Valeant Pharmaceuticals, Costa Mesa, CA) in 10 µl was added to each well. The cells were harvested 12 h later, and thymidine incorporation was measured using a Betaplate scintillation counter (Tomtec, Hamden, CT). Each in vitro stimulation was performed in quadruplicate. Supernatants were harvested after 96 h for cytokine analysis.
Phenotypic and intracellular staining of T cells
Cell surface molecules were analyzed by FACS. After blocking FcRs with excess anti-Fc (24.G2), cells were incubated with fluorescently conjugated Abs against CD4 (FITC or allophycocyanin), CD3 (PE), CD8 (FITC), OX40 (PE), CD25 (PE), or CD69 (PerCP). Flow cytometric measurement of cytokine production in T cells was performed according to standard protocols, with some modifications. Briefly, after lysing RBCs, PBLN or splenocytes were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (Omega Scientific), 1% L-glutamine (Invitrogen Life Technologies), 100 µg/ml streptomycin, 100 U/ml penicillin, and 50 µM 2-ME (Sigma-Aldrich). Lung cells (8 x 105 cells/well) or lymph node cells (2 x 105 cells/well) were plated in round-bottom, 96-well microtiter plates in 200 µl with increasing concentrations of OVA (500 µg/ml) for 90 h at 37°C. GolgiPlug (BD Biosciences) was added to the cultures according to the manufacturer’s instructions, and the incubation was continued for 8 h. At the end of the culture, cells were harvested, washed twice in PBS plus 2% BSA buffer, stained anti-CD4, and followed by fixation with Cytofix-Cytosperm (BD Biosciences) for 20 min at 4°C. Fixed cells were washed twice in 1x BD Perm/Wash solution (BD Biosciences) and subjected to intracellular cytokine staining in Perm/Wash buffer for 30 min at 4°C. Anti-IL-2 (FITC and PE), anti-IL-4 (PE and allophycocyanin), anti-IL-5 (PE and allophycocyanin), and anti-IFN- (FITC and allophycocyanin) were all obtained from BD Pharmingen (San Diego, CA) and used at a 1/50 dilution. Samples were analyzed for their proportion of cytoplasmic cytokines after gating on CD4+ T cells by FACSCalibur flow cytometer using CellQuest (BD Biosciences) and FlowJo software (Tree Star, San Carlos, CA).
In vitro T cell differentiation
CD4+ T cells were purified from spleen and lymph nodes by passing over nylon columns, followed by complement lysis using Abs to CD8 (3.155), heat-stable Ag (J11D), class II MHC (M5/114, Y17, and CA-4.A12), B cells (RA3.6B2), macrophage (M1/70), NK cells (PK136), and dendritic cells (33D1). T cells (5 x 105 cells/ml) were stimulated with plate-bound anti-CD3 (145-2C11; 5 µg/ml) and soluble anti-CD28 (37N51; 5 µg/ml) with or without IL-2 (30 ng/ml). After 7 days, live cells were restimulated with anti-CD3 or anti-CD3/CD28, and supernatants were harvested 24 h after secondary stimulation for assay of cytokine content by ELISA.
Statistics
Statistical significance was analyzed by Student’s t test. Unless otherwise indicated, data represent the mean ± SD, with p < 0.05 considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-deficient mice exhibit reduced lung inflammation driven by Th2 cytokines
AHR is a cardinal feature of a pulmonary allergic response and has been linked with the development of Th2 cells. To determine whether a deficiency in PKC has a direct effect on the development of airway dysfunction, wt (PKC+/+) and PKC-deficient (PKC–/–) mice were sensitized with OVA in alum to generate a Th2 response and subsequently challenged by inhalation of OVA several weeks later. Wild-type mice developed AHR in response to methacholine challenge. In contrast, PKC–/– mice showed attenuation in methacholine sensitivity, as characterized by a reduction in AHR (Fig. 1A). Airway reactivity to methacholine reflects a combination of increased smooth muscle sensitivity, due to inflammatory mediators such as histamine and leukotrienes, and airway narrowing, due to inflammation (16). To assess whether PKC deficiency influences airway inflammation, lung lavages were performed on mice killed 24 h after the last Ag challenge, and the BAL was examined for influx of total leukocytes. In OVA-primed PKC+/+ mice, the total number of BAL leukocytes dramatically increased after repeated exposure to OVA, compared with unprimed challenged mice, which was characterized by a predominant population of eosinophils (Fig. 1B). In contrast, the number of total leukocytes, including eosinophils and lymphocytes, was reduced by 
70% in the airways of PKC–/– mice.
|
AHR, eosinophila, and mucus production are controlled by Th2 cytokines, such as IL-4, IL-5, and IL-13 (17, 18, 19, 20). To determine whether the absence of PKC impaired Th2 cytokine production in the lungs, BAL was assessed for IL-4, IL-5, and IL-13 24 h after the last OVA aerosol challenge. In PKC–/– animals, the levels of IL-4 (Fig. 1I), IL-5 (Fig. 1J), and IL-13 (Fig. 1K) were reduced by an average of 60, 82, and 73%, respectively, compared with wt controls. IFN-, which was induced at only low levels above background, was not significantly different in PKC–/– mice (Fig. 1L). Because we found no evidence for elevated levels of IFN- or neutrophilia (not shown), which can accompany production of this cytokine (21), this suggests that there was not strong skewing to development of Th1 cells when PKC was absent, which is of significance when considering the results below. Collectively, these results show that PKC was required for promoting the asthmatic lung inflammation driven by Th2 cells.
PKC-deficient mice are largely unimpaired in generating lung inflammation driven by Th1 cytokines
To determine whether PKC also has a critical role in Th1 responses that result in lung inflammation, wt (PKC+/+) and PKC-deficient (PKC–/–) mice were sensitized with OVA in CFA to generate a Th1 response and subsequently challenged by inhalation of OVA several weeks later. In contrast to the Th2 response, lung inflammation driven by Th1 cells involves neutrophils, rather than eosinophils, and little/no AHR or mucus production (Fig. 2 and data not shown) (21). As demonstrated in Fig. 2, A, C, and E, wt mice developed a prominent inflammatory infiltrate that was somewhat more dispersed than in the Th2 response, but was still largely in perivascular and peribronchiolar areas. In contrast to the results with Th2-driven inflammation, lung sections from PKC–/– mice had similar infiltration to that observed in wt mice (Fig. 2, B, D, and E). Analyses of BAL showed marked and similar elevations in total leukocyte numbers in both wt and PKC–/– mice and almost identical numbers of lymphocytes, although a statistically significant reduction in neutrophils was consistently evident in the absence of PKC (Fig. 2F). Again, in contrast to the Th2 response, the signature cytokine of the Th1 response, IFN-, was not reduced in PKC–/– BAL compared with wt mice (Fig. 2G). These data show that PKC did not play a dominant role in promoting lung inflammation under the recall Th1-priming conditions used.
|
controls accumulation of Ag-induced CD4 cells during Th2, but not Th1, lung inflammation
The data from lung analyses suggested that priming of Th2 cells, but not Th1 cells, might be controlled by PKC. In the absence of functional reagents to allow tracking of OVA-reactive CD4 cells in PKC–/– mice, we performed a number of phenotypic analyses to quantitate the extent of T cell priming that accompanied the different lung inflammatory responses (Fig. 3). In the Th2 response, analyses of percentages of CD4 cells in the CD3 compartment in lung draining lymph nodes at the time of inflammation showed no difference in wt mice after Ag priming and airway challenge (Fig. 3A, top). However, when converted into total CD4 T cell numbers, a great elevation was observed in wt mice compared with nonprimed mice, but a much more modest increase was observed in PKC–/– mice (Fig. 3A, top right). Analyses of CD44 expression, a marker of Ag experience, on CD4 cells showed a significantly greater percentage of high expressors in wt mice which translated into far more primed T cells than in PKC–/– mice (Fig. 3A, middle). Lastly, analyses of OX40-expressing cells within the CD4/CD44high population also showed a marked reduction in the number induced in PKC–/– mice after Ag priming (Fig. 3A, bottom). OX40 is an inducible costimulatory molecule that we have previously shown to be a good marker of Ag-responding T cells (22). It is involved in both Th2 (22) and Th1 (S. Salek and M. Croft, unpublished observations) lung inflammatory reactions. Collectively, these data suggest that under the conditions that resulted in Th2 cytokine-driven inflammation, there were far fewer Ag-induced CD4 cells present during the lung response when PKC was absent.
|
To further delineate the extent of Th2 and Th1 priming during the opposing lung responses, we performed in vitro recall responses, restimulating draining lymph node T cells with OVA. As is typical of these divergent responses, Th2 cells in wt mice proliferated well, whereas Th1 cells displayed less proliferation based on a dose response (Fig. 4A). Proliferative responses from PKC–/– mice were strongly reduced compared with wt mice in the Th2 response, whereas they were minimally impaired in the Th1 response (Fig. 4A, left and right, respectively). Similarly, analyses of cytokine production by intracellular staining showed a marked reduction in the percentage of IL-4-producing (p < 0.05) and IL-5-producing (p < 0.01) CD4 cells in PKC–/– mice, but no difference or even enhanced percentages of IFN--producing cells (Fig. 4B). Similar results were obtained when supernatants were examined for cytokine production by ELISA (data not shown). Thus, the combined data from Figs. 3 and 4 suggested that at the time of the Th2 lung inflammatory response, fewer Ag-reactive T cells accumulated, and they displayed a partial hyporesponsive phenotype in the absence of PKC. In contrast, at the time of the Th1 lung inflammatory response, there was little evidence for defective T cell priming with normal numbers of Ag-reactive T cells that were capable of making IFN-.
|
has an early role in Th1 as well as Th2 responses
Because PKC has been thought to control initial T cell activation, and the lung inflammatory responses were induced by Ag rechallenge of mice several weeks after immunization, we assessed earlier phenotypic and functional markers of priming to determine whether the defect in Th2 responses was a result of impaired early priming, and whether any defect might be evident in the Th1 response. Analyses were conducted on draining lymph node T lymphocytes similar to those during the lung response (see Fig. 3), but on days 3 and 7 after immunization, molecules associated with T cell activation, CD25, CD69, and OX40 (Fig. 5) and production of cytokines (Fig. 6) were assessed. The number of CD4/CD44high-expressing cells that were positive for CD25, CD69, or OX40 was strongly reduced in PKC–/– mice under Th2-priming conditions even by day 3, and this phenotype was maintained through to day 7 (Fig. 5). Expression levels of these molecules on the few PKC–/– T cells that were positive were fairly comparable to those on wt T cells (data not shown), suggesting that a small number of Th2 cells was responding normally.
|
|
. This was substantiated by analyzing production of Th1 and Th2 cytokines at these time points (Fig. 6). IL-2 (p < 0.046), IFN- (p < 0.034), and IL-5 (p < 0.013) production were impaired on day 3, whereas by day 7, IL-2 and IFN- were equivalently produced in the absence of PKC.
To extend these data, we performed an in vitro differentiation assay under neutral conditions, where wt vs PKC–/– CD4 cells were stimulated over a period of 1 wk with anti-CD3 and anti-CD28 in the presence or the absence of IL-2 (Fig. 7). Upon restimulation of equivalent numbers of effector T cells generated with CD3/CD28 as well as a pronounced defect in IL-4 production, PKC–/– T cells were also impaired in secreting IFN-, albeit to a lesser extent. Addition of IL-2 largely overcame the IFN- defect, but not the IL-4 defect, supporting the idea that the strength of signaling might compensate for a dominant role for PKC in Th1 differentiation. Thus, PKC is not simply involved in Th2 responses, but can play a role in some aspects of the Th1 response and, based on our in vivo and in vitro data, can control the kinetics of differentiation into the Th1 phenotype as well as the kinetics of accumulation of Th1 cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is integral to the development of the Th2 response that results in asthma-like lung inflammation, whereas Th1 cells that infiltrate and initiate lung inflammation do not exhibit a profound requirement for PKC to be expressed. In contrast, during primary responses, both the number of T cells generated under Th1 and Th2 inflammatory conditions and the production of Th1 and Th2 cytokines were reduced in the absence of PKC, suggesting that a role in Th1 responses might be subdominant and overcome with time and/or depending on the level of inflammation. These results extend many in vitro findings on the importance of PKC to T cell priming, but also highlight the fact that certain T cell responses can develop relatively normally in the absence of this enzyme.
Many previous reports from in vitro systems have suggested that T cell activation is dependent on PKC (reviewed in Ref.1); in particular, PKC-deficient T cells were almost completely impaired in expressing CD25 and CD69 and proliferating in response to anti-CD3 and anti-CD28 (4). Additionally, a compromised in vitro recall proliferative response was reported in the latter study when assayed 2 wk after immunization of PKC-deficient mice with keyhole limpet hemocyanin in alum (4). Although our data assessing primary Th1 and Th2 responses correlated with these earlier reports on the importance of PKC, it is very interesting that lung inflammation induced by recall challenge of mice primed under Th1-polarizing conditions was only mildly affected in its absence. There was little evidence of a long term impairment of Th1 development, even though analysis of primary responses showed reduced accumulation of Th1 cells and delayed kinetics of differentiation into the Th1 phenotype. Similarly, recent data from Berg-Brown et al. (14) also showed that CTL responses to LCMV infection and CD4-dependent Ab responses to VSV infection were not reduced in PKC-deficient mice. In contrast, Berg-Brown et al. (14) did show strongly impaired CD8 responses and a form of CTL anergy in the absence of PKC when soluble peptide Ag was administered in vivo. While this manuscript was in the process of revision, Marsland et al. (23) reported defects in Th2-controlled lung inflammation in PKC-deficient mice similar to those reported in this study, after either immunization with OVA or infection with Nippostrongylus brasiliensis. Furthermore, they demonstrated that PKC-deficient mice on both the C57BL/6 background and the susceptible BALB/c background were able to mount a normal protective Th1 response against Leishmania major infection. This is also in agreement with our data showing that Th1 recall responses in the lung were minimally impaired in PKC-deficient mice that were immunized and subsequently challenged via the airways with OVA. The main difference in our data is the finding that initial priming of Th1 cells was defective, whereas these investigators did not determine whether early in vivo priming of Th1 cells was also compromised in PKC-deficient mice infected with L. major. Thus, PKC appears to have a subtler role in controlling T cell responses than previously appreciated.
Why, then, does PKC appear to be essential for some T cell responses and not others? One possibility is a differential role in CD4 vs CD8 T cells, but our data combined with those reported by Marsland et al. (23) and Berg-Brown et al. (14) argue against this. Another idea promoted by the combined results is that Th1 and Th2 cells or the generation of Th1 and Th2 phenotypes are differentially controlled by PKC. The finding of normal IgM and IgG responses to VSV, which induces Th1 immunity, are in line with this, and the finding of normal CTL responses to LCMV also loosely correlates with this idea, because such responses are type 1 (cytotoxic T cell (Tc1) and Th1) cytokine dominated. However, it needs to be tested whether every Th2- or Tc2-driven response will be critically dependent on PKC, and whether other Th1/Tc1-controlled responses can occur in its absence, before any rigid conclusion can be drawn. Additionally, it needs to be determined molecularly whether molecules such as GATA-3, c-Maf, and T-bet, which control transcription of Th2 and Th1 cytokines, are differentially controlled by PKC to fully understand the significance of the in vivo phenotypes.
In contrast to differential control of subsets of T cells, another idea regarding what dictates the use of PKC is that it is controlled by either the participation of costimulatory molecules such as CD28 or it is determined by the overall level of inflammatory signals, perhaps bridging innate and adaptive immunities, that are encountered during naive T cell priming. The latter could include production of cytokines such as IL-12 that drive Th1/Tc1 generation or cytokines using receptors with the common -chain that can control the extent of T cell clonal expansion.
The possibility that the participation of CD28 in a T cell response might be crucial to the involvement of PKC is supported by previous in vitro data centrally linking it to CD28 signals (8, 24, 25) and data showing that PKC-deficient CD8 cells behave almost identically to CD28-deficient CD8 cells (14). In relation to our work in this study and that of Marsland et al. (23) showing defective Th2-driven lung inflammation, the connection is further supported by earlier studies demonstrating that CD28/B7 interactions are also critical to similar lung responses controlled by Th2 cells (26, 27, 28). However, other data appear to argue against the simple concept that only CD28 activates PKC, and hence, a role for PKC is apparent only when CD28 is engaged. Although we do not know whether CD28 is required for the Th1 lung inflammatory response in our studies, we have unpublished data showing the involvement of two other costimulatory molecules in this response, namely OX40 and 4-1BB. Both molecules strongly synergize with CD28 (29, 30), so it is likely that CD28 may be engaged in this study even though little or no role for PKC was ultimately found in the lung response. Additionally, previous studies have shown a crucial role for CD28 in Ab production after VSV infection (31), which contrasts strongly with the unimpaired responses observed in PKC-deficient mice (14). Thus, perhaps the overall level of signaling at the time of naive T cell encounter with Ag is more crucial to whether PKC plays a dominant or a subdominant role in T cell priming rather than the involvement of CD28. Again, this might be dictated by the presence or the absence of inflammatory cytokines such as IL-12, which could dictate not only the threshold level of signals, but also the Th1/Th2 balance. In the responses where PKC has a dominant role (to soluble peptide, immunization in alum, and Nippostrongylus), there are probably major differences in the quantity and type of innate cell-derived cytokines produced compared with those responses where PKC has little apparent role (to LCMV and VSV, immunization in CFA, and Leishmania). Our data, showing that a Th1 defect can be overcome, even though the initial differentiation and accumulation of Th1 cells are impaired in the absence of PKC, are in line with the idea that the strength or duration of signaling might determine the requirement for PKC.
In summary, the results in this study show that PKC can play roles in the initial priming of Th1 and Th2 cells, but that it can have a restricted role in T cell responses as they develop with an apparent bias to being far more essential for Th2 than for Th1 inflammatory reactions. Analysis of PKC involvement in other diseases states that are driven by Th1 cells, and understanding which membrane-expressed cytokine and costimulatory receptors activate PKC and which might activate alternate pathways that bypass a requirement for PKC will be important for determining whether targeting PKC has only a limited therapeutic potential for immune diseases.
|
Acknowledgments |
|---|
-deficient mice. |
Footnotes |
|---|
1 This work was supported by National Institutes of Health Grant AI50498 (to M.C.) and CA35299 and AI49888 (to A.A.). This is manuscript 589 from the La Jolla Institute for Allergy and Immunology. 
2 A.A. and M.C. share senior authorship.
3 Address correspondence and reprint requests to Dr. Michael Croft, La Jolla Institute for Allergy and Immunology, Division of Molecular Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: mick{at}liai.org
4 Abbreviations used in this paper: PKC, protein kinase C; AHR, airway hyper-reactivity; BAL, bronchoalveolar lavage; LCMV, lymphocytic choriomeningitis virus; PAS, periodic acid-Schiff; PBLN, peribronchial lymph node; VSV, vesicular stomatitis virus; wt, wild-type mice; Tc, cytotoxic T cells.
Received for publication November 5, 2003. Accepted for publication September 3, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in T cell activation. Annu. Rev. Immunol. 20:761.[Medline]
: signaling from the center of the T-cell synapse. Curr. Opin. Immunol. 14:323.[Medline]
during T-cell activation. Nature 385:83.[Medline]
is required for TCR-induced NF-B activation in mature but not immature T lymphocytes. Nature 404:402.[Medline]
is required for TCR-induced T cell activation. Immunity 12:151.[Medline]
to membrane rafts is required for T cell activation. Nat. Immunol. 2:556.[Medline]
isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol. Cell. Biol. 16:1842.[Abstract]
B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-. Proc. Natl. Acad. Sci. USA 97:3394. participates in NF-B activation induced by CD3-CD28 costimulation through selective activation of IB kinase 
. Mol. Cell. Biol. 20:2933., a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells. Eur. J. Immunol. 29:132.[Medline]
to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
to mediate IL-4 gene expression in response to CD28 costimulation in T cells. J. Immunol. 164:3829. affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells. J. Exp. Med. 197:1525. signals activation versus tolerance in vivo. J. Exp. Med. 199:743. is critical for the development of in vivo T helper (Th)2 cell but not Th1 cell responses. J. Exp. Med. 200:181.B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes. J. Immunol. 165:1743. within the immunologic synapse. Proc. Natl. Acad. Sci. USA 99:9369.This article has been cited by other articles:
|
|
 |
|
  Y. Zhao, C. De Trez, R. Flynn, C. F. Ware, M. Croft, and S. Salek-Ardakani The Adaptor Molecule MyD88 Directly Promotes CD8 T Cell Responses to Vaccinia Virus J. Immunol., May 15, 2009; 182(10): 6278 - 6286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Salek-Ardakani, R. Arens, R. Flynn, A. Sette, S. P. Schoenberger, and M. Croft Preferential Use of B7.2 and Not B7.1 in Priming of Vaccinia Virus-Specific CD8 T Cells J. Immunol., March 1, 2009; 182(5): 2909 - 2918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Bogaert, K. G. Tournoy, T. Naessens, and J. Grooten Where Asthma and Hypersensitivity Pneumonitis Meet and Differ: Noneosinophilic Severe Asthma Am. J. Pathol., January 1, 2009; 174(1): 3 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Salek-Ardakani, M. Moutaftsi, S. Crotty, A. Sette, and M. Croft OX40 Drives Protective Vaccinia Virus-Specific CD8 T Cells J. Immunol., December 1, 2008; 181(11): 7969 - 7976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Manicassamy, D. Yin, Z. Zhang, L. L. Molinero, M.-L. Alegre, and Z. Sun A Critical Role for Protein Kinase C-{theta}-Mediated T Cell Survival in Cardiac Allograft Rejection J. Immunol., July 1, 2008; 181(1): 513 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sakowicz-Burkiewicz, G. Nishanth, U. Helmuth, K. Drogemuller, D. H. Busch, O. Utermohlen, M. Naumann, M. Deckert, and D. Schluter Protein Kinase C-{theta} Critically Regulates the Proliferation and Survival of Pathogen-Specific T Cells in Murine Listeriosis J. Immunol., April 15, 2008; 180(8): 5601 - 5612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Marsland, C. Nembrini, K. Grun, R. Reissmann, M. Kurrer, C. Leipner, and M. Kopf TLR Ligands Act Directly upon T Cells to Restore Proliferation in the Absence of Protein Kinase C-{theta} Signaling and Promote Autoimmune Myocarditis J. Immunol., March 15, 2007; 178(6): 3466 - 3473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Manicassamy and Z. Sun The Critical Role of Protein Kinase C-{theta} in Fas/Fas Ligand-Mediated Apoptosis J. Immunol., January 1, 2007; 178(1): 312 - 319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Healy, E. Izmailova, M. Fitzgerald, R. Walker, M. Hattersley, M. Silva, E. Siebert, J. Terkelsen, D. Picarella, M. D. Pickard, et al. PKC-{theta}-Deficient Mice Are Protected from Th1-Dependent Antigen-Induced Arthritis J. Immunol., August 1, 2006; 177(3): 1886 - 1893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-L. Tan, J. Zhao, C. Bi, X. C. Chen, D. L. Hepburn, J. Wang, J. D. Sedgwick, S. R. Chintalacharuvu, and S. Na Resistance to Experimental Autoimmune Encephalomyelitis and Impaired IL-17 Production in Protein Kinase C{theta}-Deficient Mice. J. Immunol., March 1, 2006; 176(5): 2872 - 2879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. N. Georas, J. Guo, U. De Fanis, and V. Casolaro T-helper cell type-2 regulation in allergic disease Eur. Respir. J., December 1, 2005; 26(6): 1119 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Salek-Ardakani, T. So, B. S. Halteman, A. Altman, and M. Croft Protein Kinase C{theta} Controls Th1 Cells in Experimental Autoimmune Encephalomyelitis J. Immunol., December 1, 2005; 175(11): 7635 - 7641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. de Souza, T. B. Oriss, K. J. O'Malley, A. Ray, and L. P. Kane T cell Ig and mucin 1 (TIM-1) is expressed on in vivo-activated T cells and provides a costimulatory signal for T cell activation PNAS, November 22, 2005; 102(47): 17113 - 17118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Barouch-Bentov, E. E. Lemmens, J. Hu, E. M. Janssen, N. M. Droin, J. Song, S. P. Schoenberger, and A. Altman Protein Kinase C-{theta} Is an Early Survival Factor Required for Differentiation of Effector CD8+ T Cells J. Immunol., October 15, 2005; 175(8): 5126 - 5134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tanaka, T. So, S. Lebedeva, M. Croft, and A. Altman Impaired IL-4 and c-Maf expression and enhanced Th1-cell development in Vav1-deficient mice Blood, August 15, 2005; 106(4): 1286 - 1295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Martin, R. Villares, S. Rodriguez-Mascarenhas, A. Zaballos, M. Leitges, J. Kovac, I. Sizing, P. Rennert, G. Marquez, C. Martinez-A, et al. Control of T helper 2 cell function and allergic airway inflammation by PKC{zeta} PNAS, July 12, 2005; 102(28): 9866 - 9871. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Cells stained with isotype-matched control mAb. The numbers in each histogram indicate the percentage of CD3+CD4+ cells that express high levels of CD44. Bottom panel, Percentage (left) and absolute numbers (right) of OX40+ cells gating on CD4+CD44high T cells. Quadrant settings, distinguishing positive from background fluorescence, were determined by staining with isotype-matched control mAbs (not shown). *, p < 0.0001 (vs wt mice immunized and challenged with OVA). Similar results were obtained in two independent experiments.
; n = 4) and PKC+/+ (•; n = 4) mice were tested in vitro for T cell reactivity to OVA. A, Proliferation in lung draining lymph nodes after 96 h of stimulation with increasing concentrations of OVA as indicated. Results are the mean ± SEM from quadruplicate cultures, using cells pooled from four to seven mice per group. B, Lung draining lymph node cells were restimulated in vitro with OVA for 96 h. Cells were harvested and stained for CD4 and intracellular IL-4, IL-5, or IFN-
