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-Deficient Mice Are Protected from Th1-Dependent Antigen-Induced Arthritis
* Inflammation Department and
Imaging Sciences, Millennium Pharmaceuticals, Cambridge, MA 02139
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Abstract |
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transduces the signal from the TCR through activation of transcription factors NF-
B, AP-1, and NFAT. We examined the effects of PKC- deficiency on two Th1-dependent models of Ag-induced arthritis and found that PKC--deficient mice develop disease, but at a significantly diminished severity compared with wild-type mice. In the methylated BSA model, cellular infiltrates and articular cartilage damage were mild in the PKC--deficient mice as compared with wild-type mice. Quantitation of histopathology reveals 63 and 77% reduction in overall joint destruction in two independent experiments. In the type II collagen-induced arthritis model, we observed a significant reduction in clinical scores (p < 0.01) in three independent experiments and diminished joint pathology (p < 0.005) in PKC--deficient compared with wild-type littermates. Microcomputerized tomographic imaging revealed that PKC- deficiency also protects from bone destruction. PKC--deficient CD4+ T cells show an impaired proliferative response, decreased intracellular levels of the cytokines IFN-
, IL-2, and IL-4, and significantly diminished cell surface expression of the activation markers CD25, CD69, and CD134/OX40 on memory T cells. We demonstrate decreased T-bet expression and significantly reduced IgG1 and IgG2a anti-collagen II Ab levels in PKC--deficient mice. Collectively, our results demonstrate that PKC- deficiency results in an attenuated response to Ag-induced arthritis, which is likely mediated by the reduced T cell proliferation, Th1/Th2 cell differentiation and T cell activation before and during disease peak. |
Introduction |
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is a calcium-independent isoform of the PKC family of serine-threonine kinases that relays the TCR signal for T cell activation. Upon TCR/CD28 engagement, PKC- is localized to the immunological synapse, interacts with other signaling mediators, e.g., Lck (1), and amplifies the initial TCR signal via activation of transcription factors NF-B and AP-1 (2). Activation of PKC- via TCR ligation results in IL-2 secretion, increased IL-2R expression, and the ensuing clonal expansion of CD4+ and CD8+ T cells (2, 3).
PKC- expression is largely restricted to T cells, skeletal muscle, and platelets (4). The role of PKC- in signaling the immune response combined with the restricted expression profile has focused efforts toward developing immunosuppressive therapeutics targeting PKC-. Recent studies have focused on identifying the role of PKC- in Th1- and Th2-mediated experimental diseases. Th progenitor cells differentiate into distinct T cell subsets, Th1 or Th2 cells, which are defined by the cytokine expression profile, dictate effector function, and which affect the host response to disease. Several independent studies confirm that PKC--deficient mice exhibit an impaired Th2 response in experimentally induced airway disease (3, 5, 6). However, PKC--deficient mice were resistant to Leishmania major infection, a classic model of Th1-mediated immune responses, suggesting the Th1 response was normal. Moreover PKC--deficient mice developed significant lung inflammation and expressed Th1 cytokines following an airway recall challenge in OVA-induced airway hyperresponsiveness (5, 6). Collectively the results of these studies have led investigators to conclude that PKC- function may be more relevant to Th2- rather than Th1-mediated diseases. However, the majority of these studies were focused on models of lung disease.
Ag-induced arthritis (AIA) is a well-characterized, Th1-mediated experimental model of joint disease (7). The results of a previous study investigating PKC- activity in Th1-mediated lung disease suggested that the strength of the immunogenic stimuli affects the intensity of the Th1-mediated response (6). For these reasons, we tested the effects of PKC- deficiency on the development of AIA using two models, methylated BSA (mBSA)-induced arthritis and type II collagen-induced arthritis (CIA). Methylated BSA induces a milder form of joint inflammation compared with CIA. Our results demonstrate that in two models of AIA, PKC--deficient mice are protected from joint disease. PKC--deficient, splenic T cells isolated from either mBSA or type II collagen immunized mice show an impaired proliferative response in a recall assay. CD4+ splenic T cells also exhibit reduced expression of the Th1 transcription factor T-bet (T-box expressed in T cells). CD4+ T cells isolated from draining lymph nodes (LN) reveal diminished levels of intracellular IFN- (a Th1 cytokine), IL-2, and IL-4 (a Th2 cytokine) before disease onset and during disease peak. Memory CD4+ T cells increase surface expression of CD25, CD69, and CD134/OX40 during disease progression, but these activation markers are also significantly reduced in PKC--deficient cells. Of note, is our observation that the generation of both IgG1 (Th2-dependent) and IgG2a (Th1-dependent) anti-collagen type II Ab levels are significantly decreased in PKC--deficient mice as compared with wild-type mice. These results suggest that PKC- deficiency inhibits Th cell differentiation to the Th1 and Th2 phenotype and also inhibits T cell activation. Collectively, our results demonstrate that PKC- is required to mount a T cell-dependent response in experimental joint disease.
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Materials and Methods |
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All animals were housed and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee at Millennium Pharmaceuticals. PKC--deficient mice were obtained from Dr. D. R. Littman (Skirball Institute, New York University School of Medicine, New York, NY) (2). All animals used in this study were either bred and maintained or purchased from Charles River Breeding Laboratories.
Methylated BSA-induced arthritis
C57BL/6 mice, either wild-type or containing a gene deletion in PKC- (2), were used to induce disease. Female or male mice between 8 and 10 wk of age received 200 µg of mBSA (Sigma-Aldrich) emulsified in CFA (Difco) via s.c. injection into the right flank. Seven days following the initial immunization, mice received 100 µg of mBSA in CFA via intradermal injection at the base of the tail. Twenty-one days after the initial immunization, anesthetized mice received 30 µg of mBSA in saline or saline alone via intra-articular injection into the left knee. Mice were euthanized 28 days after the initial immunization and the joints were dissected, fixed in Formalin, and prepared for histology.
The histological score was determined using a 1 through 4 scale based on the intensity and extent of change to the joint space for each of five categories. A score of 1 represents a minimal change and a score of 4 a marked change. The histological score is the sum of the five categories: inflammation, synovial alterations, cartilage degeneration, pannus formation, and bony changes. Scores were determined by a pathologist.
CIA induction
PKC--deficient mice, bred onto the DBA/1J background for four and five generations, were used in these studies. Male mice between 8 and 12 wk of age received 100 µg of bovine type II collagen (Chondrex) in CFA via s.c. injection at the base of the tail. Twenty-one days following the initial immunization, mice received 100 µg of bovine type II collagen in IFA (Difco). Clinical scores were assessed beginning on day 28 following the initial immunization. A clinical score of 0 is assigned for no visible signs of paw swelling; 0.5 for redness or slight swelling; 1.0 for obvious swelling in one or more toes; 1.5 for moderate swelling at ankle/wrist and/or moderate swelling at multiple locations; 2.0 for severe, localized swelling at the ankle; 2.5 for severe local swelling and involvement of additional locations; 3.0 for severe swelling over the whole paw with all joints affected; 3.5 for some loss in the range of motion; and 4.0 for complete ankyloses. Approximately 60 days after the initial immunization, animals were euthanized and serum and tissues were collected for additional analyses.
T cell proliferation assays
Animals received a primary immunization with either mBSA or bovine type II collagen as described. Spleens were harvested from mBSA-treated animals 21 days postimmunization and from bovine type II collagen-treated mice 35 days postimmunization. Splenocytes were dispersed using a cell strainer, and splenic T cells were isolated by negative selection using two filtrations through mouse T Cell Enrichment Columns (R&D Systems). T cells (2 x 106 cells/ml) were cocultured with mitomycin C-treated splenocytes (4 x 106 cells/ml) isolated from naive mice. Cells were grown in RPMI 1640 (Invitrogen Life Technologies) with 1% normal mouse serum in the presence of mBSA or bovine type II collagen. Cells were incubated in round-bottom, 96-well microtiter plates at 37°C for 3 days (Corning). Cells were incubated with 1 µCi of [3H]thymidine per well (MP Biomedicals) for 16 h. The cells were collected, washed and [3H]thymidine incorporation was quantified.
Microcomputerized tomographic (microCT) imaging
MicroCT imaging of Formalin-fixed mouse paws was used to measure bone surface roughness. At day 64, animals were euthanized and paws were immersed in 10% buffered Formalin for 24 h. MicroCT imaging was performed on a Scanco Medical MicroCT-40. The excised hind paws were secured in 36-mm imaging tubes containing 10% buffered Formalin. Approximately 200–400 slices, with 18.8-mm slice thickness, were acquired on a 1024 x 1024 image matrix with digital resolution of 18 µm x 18 µm x 18.8 µm. Imaging parameters included a 175 ms exposure time, 70 kVp, 114 mA, and 2000 projections over 360° angles.
Raw microCT data were imported into the software package Analyze (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN; AnalyzeDirect). Background and soft tissue were segmented from the bone in the images using histogram analysis. By this method, any portion of the tibia superior to the lateral malleolus was digitally removed. Finally, the data were linearly interpolated to an isotropic voxel dimension of 36 µm.
Roughness analysis was performed as previously described (8). Briefly, a threshold was set for the three-dimensional microCT data and the remaining objects (i.e., the bones of the foot) were converted from the volumetric (voxel) data to a geometric surface mesh and each part of the surface was analyzed with respect to its immediate neighborhood. Within a surface neighborhood (a circular region of 
500 µm), the average roughness was calculated and converted into an image. A histogram of roughness for the entire surface was generated. Using the histogram of control (nondiseased) samples, a threshold was calculated delineating smooth from rough regions. This critical step lessens the influence of normal form and waviness that is present on the complicated bone surface structure of a mouse foot. For each sample, the degree of roughness greater than the threshold was summed and a roughness score assigned to each bone sample.
Flow cytometry
The conjugated Abs were obtained from BD Biosciences (unless otherwise stated): anti-mouse CD4-FITC, anti-mouse CD44-PE, anti-mouse CD25-PerCP, anti-mouse CD69-PerCP, and anti-mouse CD134-allophycocyanin (Serotec). Ab concentrations were used according to the manufacturer’s protocol. FACS was performed using freshly isolated or cultured cells from inguinal LN in a 96-well format. Cells were blocked with an excess of anti-Fc receptors and incubated with Abs for 15 min at room temperature and washed twice with PBS containing 1% BSA (Sigma-Aldrich). Cells were fixed with BD FACSLysis solution (BD Biosciences).
Intracellular staining for IFN-, IL-2, and IL-4 was performed as described previously (9) using the following Abs: anti-IFN- (PE), anti-IL-4 (PE), anti-IL-2 (PE), and anti-mouse CD4 (allophycocyanin). Samples were analyzed by flow cytometry using a FACSCalibur (BD Biosciences).
Quantitative RT-PCR
Splenocytes were dispersed using a cell strainer and CD4+ T cells were separated by negative selection using mouse CD4+ Enrichment Columns (R&D Systems). T cells were pelleted and RNA was isolated using the Qiagen Mini Prep kit. cDNA was generated using TaqMan reverse transcription reagents (Applied Biosystems) following the manufacturer’s instructions. The reaction mix contained 1x reverse-transcribed buffer, 5.5 mM MgCl2, 2 mM dNTPs, 50 µM random hexamers, 2.5 mM oligo(dT), 40 U RNase inhibitors, 125 U Multiscribe reverse transcription, and RNA in a 100-µl volume. cDNA was diluted 1/10 in H2O and stored at 4°C. Quantitative RT-PCR (TaqMan) was performed on ABI PRISM 7700 apparatus (Applied Biosystems) as previously described (10). The sequences used to amplify T-bet were the primers forward 5'-CCTGTTCCCAGCCGTTTCTAC-3', reverse 5'-AGCCAGTAAGGCTGTGAGATCATATC-3' and the probe 5'-CGACCTTCCAGGCCAGCCCAA-3'. The sequences used to amplify IFN- were forward 5'-CAGCACTCGAATGTGTCAGGTAGT-3', reverse 5'-GGAAGACCAGTGTCAAGTCTCTTG-3' and the probe 5'-GACACTGCTTTCTTTCAGGGACAGCCTGT-3'. The amplification protocol was step 1, 50°C for 2 min, 95°C for 10 min; and step 2 for 40 cycles, 95°C for 15 s, 60°C for 1 min.
ELISA
Serum levels of mouse anti-type II collagen total IgG, IgG1, and IgG2a were measured. Plates were coated with mouse type II collagen (Chondrex) at 4 µg/ml in PBS (pH 7.2–7.4) containing 0.05% Tween 20 (assay buffer) at 4°C overnight. Nonspecific binding to the plate surface was blocked by incubating plates with assay buffer containing 1% goat serum at room temperature for 2 h. Serial dilutions of the immune and nonimmune or control serum were added to the assay buffer and incubated at room temperature for 1 h. The HRP-conjugated secondary Ab was diluted 1/25,000 to detect total IgG (RDI) or 1/1000 to detect either anti-IgG1 or anti-IgG2a (BD Biosciences) before incubation at room temperature for 1 h. The enzyme-substrate reaction was conducted using tetramethylbenzidine peroxidase substrate (TMB; Kirkegaard & Perry Laboratories) and the reaction was stopped with TMB solution (Kirkegaard & Perry Laboratories).
Statistical analyses
For the CIA model, a repeated-measures linear model was used to compare knockout and wild-type animals with respect to their pattern of clinical scores over time. The model included the day of the experiment, the type of animal (knockout or wild type), and the interaction between these two terms, with variable of interest being the interaction term. A spatial power covariance structure was assigned to the model to describe the correlation between the multiple measurements within each animal. For microCT imaging, statistical significance was calculated using ANOVA with a Newman-Keuls post hoc test, all other comparisons were made using ANOVA with values for p < 0.05 considered significant.
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Results |
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deficiency attenuates soft tissue infiltration and cartilage destruction in mBSA-induced arthritis
To investigate the effects of PKC- deficiency on the development of AIA, we first compared wild-type and knockout mice in the mBSA model of arthritis. To determine the extent of disease activity, we analyzed joint histology and observed that sections through the knee of wild-type mice reveal marked inflammation in the soft tissue of the infrapatellar fat pad and loss of cartilage at the articular surface. In contrast, the PKC--deficient knees appeared less inflamed (Fig. 1, compare B with D), with no detectable loss of articular cartilage (detected by toluidine blue staining) (data not shown). To quantify these differences, we used a histopathological scoring system. In two independent studies, the majority of PKC- wild-type mice (n = 6 of 8 and 8 of 8, respectively) developed cellular infiltrates, synovial alterations, and either osteolysis/effacement or periosteal reaction. PKC--deficient mice also exhibited cellular infiltrates and synovial alterations (n = 7 of 8 and 7 of 10, respectively) but these were markedly reduced and there was no evidence for osteolysis or periosteal reaction. The pathological changes are reflected in the total pathology score and represent a 63 and 77% reduction from wild-type levels (Fig. 2).
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deficiency attenuates disease in CIA
The marked protection against soft tissue inflammation and cartilage degradation observed in the mBSA-induced arthritis model led us to ask whether disease was also diminished in the more destructive model of AIA, the CIA model. To test this question, we bred the PKC--deficient mice onto the CIA-susceptible mouse strain, DBA/1J, and induced disease in wild-type and knockout littermates. In three independent experiments, PKC--deficient mice exhibit lower clinical scores compared with wild-type mice and the differences between the two scoring trends within each experiment are statistically significant (Fig. 3). By day 64, disease is less severe in PKC--deficient animals, and we note a delay in disease onset. To address whether the diminished disease severity observed in PKC--deficient mice reflected solely a delay in disease onset, in one of three experiments, we continued to score animals through day 148. In this study we found a similar profile for the clinical score observed by day 64; that is PKC--deficient mice develop a milder disease that does not develop to wild-type levels even after 148 days (Fig. 3, experiment 3). Furthermore, we observe a significant decrease in the histopathological score in knockout compared with wild-type mice (Fig. 4).
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and IL-17, contribute directly to bone destruction in rheumatoid arthritis (RA) by increasing osteoclast activity (11). To address whether PKC- deficiency affects bone destruction in the CIA model, we quantified bone erosion using microCT imaging bone surface roughness analysis. Bone surface roughness increases as a result of the highly erosive nature of arthritis (8). When comparing the wild-type and knockout paws from animals with arthritis, we observe the roughness score for the knockout animals is identical with control animals without disease. However, the wild-type animals subjected to CIA have a 2.5-fold increase in bone surface roughness as compared with controls (Figs. 5 and 6).
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-deficient mice
PKC--deficient T cells show an impaired proliferative response following cross-linking of the TCR (2). To increase our understanding of the impaired arthritic response to Ag, we isolated splenocytes from mBSA or type II collagen-immunized wild-type and PKC--deficient mice and measured T cell proliferation in a recall response. T cells isolated from either mBSA or type II collagen-immunized mice proliferate in culture as determined by increased [3H]thymidine incorporation. In all experiments, T cells deficient in PKC- proliferate in response to Ag but at significantly lower levels than wild-type T cells (Fig. 7). However, T cells from wild-type and PKC--deficient mice responded equally to the T cell mitogen Con A (data not shown).
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-deficient mice exhibit a diminished Th1 phenotype in secondary lymphoid organs before disease onset
To delineate the molecular mechanisms resulting in diminished joint disease, we isolated lymphocytes from the draining inguinal LN and measured intracellular cytokine expression and cell activation markers by flow cytometry. CD4+ LN T cells maintained in culture with mBSA for 72 h express IFN-, IL-2, and IL-4 at both 14 and 28 days postimmunization. In wild-type mice the percentage of CD4+ LN T cells expressing IFN- decreases from day 14 (26 ± 3.8%) to day 28 (7.5 ± 1.2%); the percentage of CD4+ LN T cells expressing IL-2 (5.2 ± 1%) and IL-4 (12 ± 1%) remain unchanged between day 14 and day 28 postimmunization (Fig. 8a). At both time points, the percentage of CD4+ LN T cells expressing IFN-, IL-2, or IL-4 is significantly reduced in PKC--deficient mice as compared with wild-type mice (Fig. 8a). For example, the percentage of CD4+ LN T cells expressing IFN- at day 14 in PKC--deficient mice is 4.5 ± 1.6% vs 26 ± 3.8% in the wild-type mice (Fig. 8a).
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-deficient mice express significantly reduced numbers of CD25 (0.5 x 105), CD69 (0.2 x 105), and CD134/OX40 (0.69 x 105) as compared with wild-type mice.
T-bet expression increases during disease development in wild-type but not PKC--deficient mice
The results of previous studies indicate that T-bet drives expression of IFN-, committing T cells to the Th1 subset. Recent work established that T-bet protein levels correlate with disease activity in patients with aplastic anemia and that T-bet expression levels may be an indicator of disease activity (12). We compared T-bet expression in the spleens of wild-type mice and PKC--deficient mice during disease development. Freshly isolated, CD4+ enriched splenic T cells from both wild-type and PKC--deficient mice show comparable levels of T-bet expression at day 14 postimmunization. T-bet levels increase significantly in wild-type mice (3.8-fold), but not in PKC--deficient mice (2.3-fold), 28 days postimmunization. A similar pattern is seen for IFN- RNA levels, however none of the comparisons were statistically significant (Fig. 9). The distinct patterns in IFN- expression observed between the percentage of CD4+ LN T cells and CD4+ enriched splenic cells shown in Figs. 8 and 9, is likely due to the source of T cells (draining LN vs spleen), the purity of the enriched CD4+ cells (80% pure CD4+ T cells used for RT-PCR) and the methods of detection (flow cytometry on single cells vs expression analysis from a pool of total transcripts).
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-deficient mice
We next asked whether the impaired T cell response of PKC--deficient mice would affect the production of Ag-specific Abs in AIA. To accomplish this, we measured the production of anti-mouse type II collagen-specific Abs from animals with mBSA-induced arthritis on day 28 and with CIA on day 61 (Fig. 10). First, we measured total IgG. Total anti-type II collagen IgG production was reduced 7-fold in the PKC--deficient mice compared with the wild-type animals with mBSA-induced arthritis. Total anti-type II collagen IgG Ab levels were reduced 18-fold in the PKC--deficient mice compared with the PKC- wild-type animals with CIA. It is of interest to note that the production of total IgG anti-collagen Abs was not abrogated completely and was still detectable in the PKC--deficient animals in both mBSA-induced arthritis and CIA. Second, to investigate how PKC- deficiency affects the balance between Th1 and Th2 polarization, we measured the production of collagen-specific IgG2a, which is typically produced during a Th1 response, and collagen-specific IgG1, which is typically produced during a Th2 response. Ab subtypes were reduced in PKC--deficient animals with mBSA-induced arthritis; anti-collagen IgG2a was reduced 4.5-fold and anti-collagen IgG1 reduced by 10-fold. We observed a similar reduction in collagen-specific IgG2a (12.5-fold) and IgG1 (69-fold) in the PKC--deficient mice with CIA. In both AIA models, production of the anti-collagen IgG2a Abs was significantly reduced in the PKC--deficient mice, but still detectable, whereas, the anti-collagen IgG1 in PKC--deficient mice was similar to the levels of the nonimmune mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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function is coupled to TCR activation and signals the NF-B-, NFAT-, and AP-1-mediated transcription of genes whose products are required for T cell proliferation and differentiation (2, 13, 14). The results of studies performed in vitro revealed the essential role of PKC- for T cell proliferation in response to TCR engagement (reviewed in Ref. 15). The results of studies performed in vivo indicate that PKC- is required for Th2- but not Th1-dependent experimentally induced airway diseases (6). More recently, two independent groups, as well as unpublished work from our laboratory, showed that PKC--deficient mice are resistant to experimental autoimmune encephalomyelitis (EAE), a Th1-dependent disease model (6, 9, 16). The results of our studies clearly demonstrate that PKC- is required to mount a productive Th1-dependent immune response to experimentally induced arthritis in rodents. We show in this study that PKC--deficient mice exhibit decreased disease severity in two models of AIA, mBSA-induced arthritis and CIA. Several mechanisms appear to contribute to this diminished pathological phenotype. Our results indicate a reduced proliferative capacity of PKC--deficient T cells in response to Ag and decreased intracellular IL-2 levels. Expression of the transcription factor controlling Th cell lineage commitment, T-bet, is impaired and CD4+ T cells express decreased levels of the intracellular Th1 cytokine, IFN-, as well as the Th2 cytokine, IL-4. Activation of CD4+ memory T cells is reduced as evidenced by decreased expression of CD25, CD69, and CD134/OX40. Finally, we detect decreased murine anti-collagen type II Ab titers in PKC--deficient mice in response to immunization with both Ags.
The results of a previous study suggest that the extent or duration of inflammation dictates whether PKC- signaling is required to mount an effective Th1 response (6). To test this supposition, we chose to examine two distinct models of AIA. The mBSA model exhibits a mild joint pathology as compared with the CIA model, which exhibits intense edema, inflammation, and joint destruction. It has also been suggested that the requirement for PKC- function in Th1 differentiation may be time-dependent (6); however, we determined that the mechanism of reduced disease is not a result of delayed onset. Finally, the more destructive disease progression of the CIA model as compared with the mBSA model is evidenced not only in the clinical symptoms and joint pathology but also by microCT analysis of bone. PKC- expression is largely restricted to T cells and muscle, therefore the protective effect on bone erosion in PKC--deficient mice is secondary to the diminished joint inflammation.
Human RA is characterized by the production of a wide spectrum of autoreactive Abs. The arthritogenic potential of anti-type II collagen Abs has been well documented in experimental models (17). We demonstrate that PKC--deficient mice exhibit an impaired production of total anti-type II collagen IgG in both models of AIA. These results indicate that PKC- deficiency impairs the production of self-reactive Abs and likely contributes to disease amelioration. Decreased Ab titers may affect the rate of immune complex formation, complement fixation and the subsequent recruitment of inflammatory cells to the diseased joints. Th1 cell commitment may be impaired in PKC--deficient mice, as suggested by T-bet expression levels. To further investigate whether PKC- deficiency affected T cell lineage commitment following T cell activation, we measured the production of anti-collagen IgG2a subtype, which is produced during a Th1 response, and the IgG1 subtype, indicating a Th2 response. The production of both IgG2a and IgG1 subtypes were impaired in the PKC--deficient mice that developed either mBSA-induced arthritis or CIA. Impaired IgG2a production, in addition to decreased intracellular IFN- levels, supports the supposition that PKC- is required for Th1-mediated diseases, like RA. The deficiency in the IgG1 production is in concordance with decreased levels of intracellular IL-4 detected by flow cytometry and also with previous results indicating that PKC--mediated signaling is required to mount Th2-dependent responses. The decreased level of both IgG subtypes supports the supposition that PKC- deficiency affects commitment to both the Th1 and Th2 phenotype.
Several studies have contributed to our understanding of PKC- function in disease. The studies of Berg-Brown et al. (18) addressed the viral immune response in PKC--deficient mice. PKC- wild-type and deficient mice mounted an identical immune response to lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus, however, the cytotoxic activity of CD8 T cells in vitro in response to LCMV Ag, gp33, was impaired in PKC--deficient cells. A more recent study comparing PKC- deficiency in the CD8+ T cell response to viral exposure in vitro vs in vivo suggests that LCMV activates the innate immune response and indicates that PKC- signaling is not essential for antiviral immune responses in vivo (3). The role of PKC- in autoimmunity appears more complex. Studies focused on the role of PKC- in several models of experimentally induced airway disease revealed PKC- is required for an appropriate Th2, but not a Th1, response in vivo (5, 6). In contrast, two more recent studies (9, 16) demonstrate that PKC--deficient mice are resistant to EAE, a Th1-dependent disease. The results reported in this study demonstrate the requirement of PKC- for a Th1-dependent response to Ag in the development of joint disease. The requirement for PKC- signaling in the development of the Th1-dependent response in both the CNS and the joints but not the lung suggests that the target organ may also contribute to the type of immune response. Collectively, the results from in vivo studies demonstrate PKC- deficiency leads to an impaired response in Th1- and Th2-mediated immune challenges and suggest this response may be tissue or organ specific.
There is increasing evidence suggesting that the local cytokine profile is important for a complete and productive T cell response. Many of the cytokines that determine the T cell response are regulated by the transcription factors NF-B, AP-1, and NFAT. PKC- activates all three transcription factors (2, 14, 19, 20, 21, 22). It is interesting to note that recent results suggest that PKC--mediated NFAT activation is dependent upon the extent of TCR cross-linking, unlike NF-B and AP-1 activation, and that following TCR activation and PKC- signaling, downstream signaling events may diverge (14, 21). These results, generated from studies performed in vitro, lend support to evidence generated in vivo indicating that Ag-based differences exist in PKC- signaling.
During AIA, intracellular levels of IFN- were significantly reduced in PKC--deficient mice before disease onset and during disease peak. A similar pattern was observed for IL-2 and IL-4. The cytokine profile we observed in the AIA model is similar to the intracellular cytokine levels in CD4+ T cells isolated from the draining LNs of mice with myelin oligodendrocyte glycoprotein 35–55-induced EAE (9). Although the methods vary and the temporal pattern of cytokine expression differs between the arthritis models and the EAE model as each disease develops and peaks, the overall findings are consistent, PKC--deficient CD4+ T cells express reduced cytokine levels.
Rodent AIA models are T cell-dependent and exhibit a distinct imbalance in T cell polarization toward the Th1 phenotype. However, the role of T cells, and in specific an imbalance in Th cell polarization, in the pathogenesis of human RA remains somewhat controversial. What is established is that T cells are a component of the synovial inflammation. Furthermore, the results of several studies analyzing peripheral blood, synovial fluid, and tissue from patients with RA demonstrate a shift in the balance toward the Th1 phenotype, i.e., IFN-+/IL-4–, CD4+ T cells and/or measurable Th1 cytokines (23, 24, 25, 26). Therefore, Th1 effector cells are present and presumably contribute to human disease (27).
We show that PKC- is required to mount a productive, Th1-dependent response to AIA. Furthermore, we provide evidence suggesting there are multiple molecular mechanisms contributing to diminished joint disease. These include decreased T cell proliferation, differentiation not only to a Th1 but also the Th2 phenotype, and effector functions. Our results confirm studies demonstrating a role for PKC- in T cell proliferation and activation in vivo and extend these findings by demonstrating an imbalance in both the Th1 and Th2 compartment during AIA.
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Acknowledgments |
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knockout mice and Dr. L. Kelly-Modis for a critical reading of this manuscript. |
Disclosures |
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Footnotes |
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1 Address correspondence and reprint requests to Dr. Aileen M. Healy at the current address, Momenta Pharmaceuticals, 675 West Kendall Street, Cambridge, MA 02142. E-mail address: ahealy{at}momentapharma.com 
2 Abbreviations used in this paper: PKC, protein kinase C; CIA, collagen-induced arthritis; AIA, Ag-induced arthritis; RA, rheumatoid arthritis; mBSA, methylated BSA; LN, lymph node; EAE, experimental autoimmune encephalomyelitis; LCMV, lymphocytic choriomeningitis virus.
Received for publication November 21, 2005. Accepted for publication May 1, 2006.
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  T. Gruber, N. Hermann-Kleiter, R. Hinterleitner, F. Fresser, R. Schneider, G. Gastl, J. M. Penninger, and G. Baier PKC-{theta} Modulates the Strength of T Cell Responses by Targeting Cbl-b for Ubiquitination and Degradation Sci. Signal., June 23, 2009; 2(76): ra30 - ra30. [Abstract] [Full Text] [PDF]
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 T. Letschka, V. Kollmann, C. Pfeifhofer-Obermair, C. Lutz-Nicoladoni, G. J. Obermair, F. Fresser, M. Leitges, N. Hermann-Kleiter, S. Kaminski, and G. Baier PKC-{theta} selectively controls the adhesion-stimulating molecule Rap1 Blood, December 1, 2008; 112(12): 4617 - 4627. [Abstract] [Full Text] [PDF]
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 S. C. Morley, K. S. Weber, H. Kao, and P. M. Allen Protein Kinase C-{theta} Is Required for Efficient Positive Selection J. Immunol., October 1, 2008; 181(7): 4696 - 4708. [Abstract] [Full Text] [PDF]
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 M. Deruaz, A. Frauenschuh, A. L. Alessandri, J. M. Dias, F. M. Coelho, R. C. Russo, B. R. Ferreira, G. J. Graham, J. P. Shaw, T. N.C. Wells, et al. Ticks produce highly selective chemokine binding proteins with antiinflammatory activity J. Exp. Med., September 1, 2008; 205(9): 2019 - 2031. [Abstract] [Full Text] [PDF]
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 K. M. Page, D. Chaudhary, S. J. Goldman, and M. T. Kasaian Natural killer cells from protein kinase C {theta}-/- mice stimulated with interleukin-12 are deficient in production of interferon-{gamma} J. Leukoc. Biol., May 1, 2008; 83(5): 1267 - 1276. [Abstract] [Full Text] [PDF]
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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]
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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]
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 E. J.A van Wanrooij, G. H.M van Puijvelde, P. de Vos, H. Yagita, T. J.C. van Berkel, and J. Kuiper Interruption of the Tnfrsf4/Tnfsf4 (OX40/OX40L) Pathway Attenuates Atherogenesis in Low-Density Lipoprotein Receptor-Deficient Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 204 - 210. [Abstract] [Full Text] [PDF]
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