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The Journal of Immunology, 2003, 170: 5772-5777.
Copyright © 2003 by The American Association of Immunologists

Impaired Secretion of IL-10 by T Cells from Patients with Common Variable Immunodeficiency–Involvement of Protein Kinase A Type I1

Are Martin Holm2,*, Pål Aukrust*,{dagger}, Einar Martin Aandahl§, Fredrik Müller*,{ddagger}, Kjetil Taskén§ and Stig S. Frøland*,{dagger}

* Research Institute for Internal Medicine, {dagger} Section for Clinical Immunology and Infectious Diseases, Medical Department, and {ddagger} Department of Microbiology, National Hospital, and § Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4+/CD8+, CD45RA+/RO+, or CD28- T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Common variable immunodeficiency (CVID)3 comprises a heterogeneous group of B cell deficiency syndromes characterized by defective Ab production and recurrent sinopulmonary infections, frequently complicated by autoimmune disorders, granulomatous inflammation, and lymphoid and gastrointestinal malignancies (1). Although deficient production of Igs by B cells is the immunologic hallmark of CVID, T cell abnormalities are present in a high proportion of the patients (2, 3, 4, 5, 6, 7). The observation that B cells from some CVID patients can produce Ig if appropriately stimulated in vitro (8, 9) suggests that T cell dysfunction leading to insufficient in vivo stimulation of B cells may be of importance for the pathogenesis of the immunodeficiency in these patients.

IL-10 is a cytokine produced mainly by monocytes/macrophages and T cells (reviewed in Ref. 10). IL-10 is released late during T cell activation and has suppressive effects on monocyte as well as T cell functions (11, 12). However, IL-10 has a stimulatory effect on B cell differentiation and Ig production (13) and, in conjunction with anti-CD40, has been found to promote Ig production in vitro in B cells from CVID patients (14, 15, 16).

cAMP is an important intracellular second messenger molecule, the effect of which is mostly mediated through cAMP-dependent protein kinases (protein kinase A (PKA)) (17, 18). Through activation of PKA type I (PKAI), cAMP may completely abolish proximal signaling events in T cells after stimulation of the TCR/CD3 complex, thus inhibiting T cell proliferation and IL-2 production (19). We have recently demonstrated enhanced PKAI activation in T cells from subgroups of CVID patients, possibly contributing to the impaired T cell function in these patients (20). Several studies suggest that the cAMP/PKAI system also affects the production of IL-10 in various leukocyte subsets. However, the reports are somewhat conflicting with regard to the effects on IL-10 secretion of T cells (21, 22, 23, 24, 25).

Because of the stimulatory effects of IL-10 on B cells and the observed T cell dysfunction in a subset of patients with CVID, we wished to study the IL-10 production of T cells from CVID patients. Because the cAMP/PKAI system may be involved in IL-10 synthesis in T cells, we also wished to examine IL-10 levels in T cell supernatants in CVID patients and healthy controls using various modes of T cell activation, particularly focusing on possible modulatory effects of the cAMP/PKAI system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and controls

Twenty-one patients (see Table I) with CVID according to the diagnostic criteria of the World Health Organization expert group for primary immunodeficiencies (1) were included in the study. The patients did not have any clinically apparent infection when recruited. The samples were obtained immediately before i.v. or s.c. infusions of Ig were administered. Eighteen healthy blood donors were included as controls. Informed consent was obtained from all study subjects before inclusion. Not all experiments were performed in all individuals due to limited numbers of cells.


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  		Table I. Clinical and immunological characteristics of CVID patients

 
Isolation of CD3+ T cells

PBMC were obtained from heparinized blood by isopaque-Ficoll (Lymphoprep; Nycomed Pharma, Oslo, Norway) gradient centrifugation within 1 h after blood sampling. For leukocyte phenotyping (see Flow cytometry analyses), PBMC were cryopreserved in liquid nitrogen (26). Further isolation of CD3+ T cells by monodisperse immunomagnetic beads was performed as previously reported (27). Briefly, PBMC suspended in PBS with 0.3% BSA (Calbiochem, La Jolla, CA), were mixed with beads coated with Abs to CD14 (Dynabeads M-450 CD14; Dynal, Oslo, Norway), CD19 (Dynabeads M-459 Pan B; Dynal), and CD56 (clone B159; BD PharMingen, San Diego, CA; bound to beads precoated with rat anti-mouse IgG1; Dynal) in a cell-to-bead ratio of 1:5. Rosetting cells were removed using a magnet after 30 min, and the negatively selected cells consisted of >95% CD3+ T cells as determined by flow cytometry.

Cell cultures and protein analyses

The negatively selected CD3+ T cells were resuspended in RPMI 1640 with 2 mM L-glutamine and 25 mM HEPES buffer (RPMI 1640; Life Technologies, Paisley, U.K.) supplemented with 10% FCS, and incubated in flat-bottom 96-well plates (106/ml; 0.2 ml/well; Costar, Cambridge, MA) with or without stimulants. Optimal concentrations of stimulants were determined in preliminary experiments. Where indicated, the cells were incubated with the cAMP analogs 8-(4-chlorophentylthio)cAMP (8-CPT-cAMP; Sigma-Aldrich, St. Louis, MO) or Rp-8-bromo-cAMP-phosphorothioate (Rp-8-Br-cAMPS; BioLog Life Science, Bremen, Germany) for 30 min before stimulation. The following stimulants were used when indicated: anti-CD3 clone SpvT3b (final dilution, 40 ng/ml; Dynal), anti-CD28 clone 15E8 (final dilution, 50 ng/ml; CLB, Amsterdam, The Netherlands), PMA (final dilution, 10 ng/ml; Sigma-Aldrich), and calcium ionophore A 23187 (final dilution, 1 µg/ml; Sigma-Aldrich). When the cells were stimulated with anti-CD3, the cell surface markers were cross-linked using monodisperse immunomagnetic beads coated with sheep anti-mouse IgG (Dynal) at a cell-to-bead ratio of 1:1. Cell-free supernatants were harvested after 16 and 48 h and stored at -80°C. The cytokine levels were determined by enzyme immunoassay (IL-10: CLB; IFN-{gamma} and IL-4: ELISA DuoSet; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Flow cytometry analyses

For measurement of intracellular IL-10 accumulation, negatively selected CD3+ T cells were cultured in 96-well flat-bottom plates (106/ml, 0.2 ml/well; Costar) that had been precoated overnight with anti-CD3 Abs (clone SpvT3b, final concentration 5 µg/ml; Dynal), or PBS. Brefeldin A (final concentration 10 µg/ml; Sigma-Aldrich) was added after 43 h to promote intracellular accumulation of IL-10. After an additional 5 h, the cells were fixed in paraformaldehyde 4%, washed, and permeabilized using FACSPerm (BD Biosciences, San Jose, CA). For lymphocyte phenotyping, thawed cryopreserved PBMC were used (26). Staining was performed using FITC-conjugated anti-CD45RA and anti-CD3, PE-conjugated anti-IL-10 and anti-CD28, PerCP-conjugated anti-CD4 and anti CD8, and allophycocyanin-conjugated anti-CD3 and anti-CD8. Isotype-matched control Abs were used where indicated. All Abs for flow cytometry were purchased from BD PharMingen. Flow cytometry was performed using a FACSCalibur instrument with CellQuest software (BD Biosciences).

Statistical methods

A two-tailed Mann Whitney U test was used comparing two independent groups of individuals. Responses within the same individual were compared by the (two-tailed) Wilcoxon signed rank test for paired data. Correlations were calculated using Spearman’s signed rank test. Tests were considered significant when p < 0.05. IC50 values were calculated by logarithmic transformation and logistic regression (Prism; Graphpad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impaired IL-10 secretion by T cells from CVID patients

In unstimulated T cells, the IL-10 levels were low in both CVID patients and controls. In stimulated T cell supernatants, the maximal levels of IL-10 were found after 48 h, while the levels after 16 h were only ~10% of the maximum (data not shown). Thus, in the following, only data after stimulating T cells for 48 h are presented. As shown in Fig. 1, anti-CD3 stimulated cells from CVID patients released significantly less IL-10 compared with cells from healthy controls. Moreover, although costimulation with anti-CD28 significantly increased IL-10 levels compared with anti-CD3 alone in both CVID patients and controls, the release of IL-10 in costimulated cells still was significantly lower in CVID patients (Fig. 1). In fact, costimulated T cells from CVID patients released IL-10 at a level similar to the T cells from healthy controls stimulated with anti-CD3 alone. In contrast, when the T cells were activated with PMA and calcium ionophore, thus bypassing the TCR/CD3 and CD28 activation, we found no significant difference between CVID patients and healthy controls in released amounts of IL-10 (mean ± SEM: 233 ± 67.7 pg/ml (CVID) vs 183 ± 70.3 pg/ml (controls), p = 0.84).



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  		FIGURE 1. Levels of IL-10 in supernatants of unstimulated, anti-CD3-stimulated, and anti-CD3/anti-CD28-stimulated T cell cultures from patients with CVID (•) and healthy controls ({circ}) after 48 h. Horizontal lines indicate median values.

 
No correlation between IL-10 response and clinical features

Previous attempts have been made to classify CVID according to certain clinical features, such as age at immunodeficiency debut, splenomegaly, autoimmune manifestations, and presence of bronchiectasis. However, in this study the in vitro secretion of IL-10 by T cells was not related to any of these factors, neither as examined by correlation analyses nor by comparing of groups with or without the clinical/immunological features that are shown in Table I.

Similar IL-10 production in the CD4+/CD8+ and CD45RA+/CD45RA- T cell subsets

A variety of T cell abnormalities have previously been observed in CVID, such as decreased a CD4+-CD8+ T cell ratio, decreased numbers of CD45RA+ T cells (2, 8), and decreased expression of CD28 (28). To investigate whether the observed reduction of secreted amounts of IL-10 in CVID might reflect a change in T cell subpopulations in these patients, we analyzed the expression of various T cell surface markers in 12 of the included patients by flow cytometry. However, we found no significant correlation between the composition of the T cell subsets of the individual patients (Table I) and the level of IL-10 in T cell supernatants. Moreover, using intracellular staining for IL-10 in anti-CD3-stimulated T cells from seven CVID patients and six healthy controls, we found reduced IL-10 production in T cells from CVID, confirming findings in T cell culture supernatants (mean fluorescence intensity: 59 ± 7.0 (CVID) vs 73 ± 7.3 (controls), p = 0.055), and notably, we found similar expression of IL-10 in the CD4+ and the CD8+ T cell subsets (mean fluorescence intensity: 58 ± 6.9 (CD4+) vs 50 ± 7.3 (CD8+) in CVID patients; and 72 ± 5.6 (CD4+) vs 75 ± 10.5 (CD8+) in the controls) (Fig. 2A). Similarly, when analyzing the CD45RA+ and CD45RA- T cell subsets, there was similar expression of IL-10 in both CVID (mean fluorescence intensity: 50 ± 7.5 (CD45RA+) vs 53 ± 8.3 (CD45RA-)) and controls (76 ± 9.4 (CD45RA+) vs 68 ± 6.6 (CD45RA-)) (Fig. 2B). Taken together, this indicates that the findings in T cell cultures from CVID patients do not merely reflect altered proportions of T cell subsets.



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  		FIGURE 2. Similar intracellular concentration of IL-10 in (A) CD4+ (left graphs) and CD8+ (right graphs) T cells and in (B) CD45RA+ and CD45RA- T cells as demonstrated by flow cytometry. Data show a CVID patient (upper graphs) with a low proportion of CD4+ T cells (40% of all T cells) and of CD45RA- T cells (20% of all T cells) and a healthy control (lower graphs). Gray line indicates unstimulated T cells, filled histogram indicates T cells stimulated for 48 h with anti-CD3.

 
cAMP agonist inhibits IL-10 secretion

To address the possible role of the cAMP/PKAI system in the reduced IL-10 production in T cells from CVID patients, we first examined the effects of the cAMP agonist 8-CPT-cAMP on IL-10 secretion in patients and controls. Although there was no effect on unstimulated cells, 8-CPT-cAMP markedly down-regulated IL-10 levels in anti-CD3-stimulated T cells in a concentration-dependent manner with a similar pattern in CVID patients and controls (Fig. 3A) with no significant difference in the concentration needed to achieve a reduction to 50% of the maximum (IC50) (median IC50 (25–75th percentiles): 3.05 (2.00–15.70) µM in CVID and 5.85 (0.65–16.05) µM in controls, p = 0.91). Moreover, in T cells costimulated with anti-CD3/anti-CD28, 8-CPT-cAMP also suppressed IL-10 levels in both CVID patients and controls (Fig. 3B). However, when costimulating with anti-CD28, the IC50 concentration was significantly lower in CVID patients than in controls (median IC50 (25–75th percentiles): 0.047 (0.042–0.052) µM in CVID and 0.065 (0.049–0.116) µM in controls, p = 0.035). In contrast to the findings in anti-CD3/anti-CD28-stimulated T cells, there was no significant suppressive effect of 8-CPT-cAMP when the T cells were activated with PMA and calcium ionophore (Fig. 3C).



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  		FIGURE 3. Levels of IL-10 in supernatants of T cells activated by anti-CD3 (A), anti-CD3/anti-CD28 (B), or PMA/calcium-ionophore (C) for 48 h, in patients with CVID ({blacksquare}) and healthy controls ({square}) after addition of different concentrations of the cAMP agonist 8-CPT-cAMP. Data are given as mean values and SEM. (*), p < 0.1; *, p < 0.05; **, p < 0.01; and ***, p < 0.001. Please note varied scaling of the x-axes.

 
The cAMP antagonist Rp-8-Br-cAMPS increases IL-10 secretion by T cells

We next examined the effect of Rp-8-Br-cAMPS, a sulfur-substituted cAMP analog with a high selectivity for PKAI (29). Similar to the agonist, we found no effect on unstimulated T cells (data not shown). However, in anti-CD3-stimulated T cells, the cAMP antagonist increased IL-10 secretion in both CVID patients and controls in a concentration-dependent manner (Fig. 4A). Notably, at the lowest concentration of Rp-8-Br-cAMPS, an enhancing effect on IL-10 levels was seen only in CVID patients, achieving a normalization of the IL-10 secretion compared with anti-CD3-stimulated T cells from healthy controls. Moreover, also when T cells were costimulated with anti-CD28, the cAMP antagonist induced a concentration-dependent increase in IL-10 levels, reaching a maximum at 250 µM (Fig. 4B). Although a similar pattern was seen in CVID patients and controls, the enhancing effect was most pronounced in the CVID patients (~3.4-fold compared with a ~2.1-fold increase). Finally, and similar to the finding when using the cAMP agonist, there was no significant effect of the cAMP antagonist in neither patients nor controls when the T cells were activated with PMA and calcium ionophore (Fig. 4C).



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  		FIGURE 4. Levels of IL-10 in supernatants of T cells activated by anti-CD3 (A), anti-CD3/anti-CD28 (B), or PMA/calcium-ionophore (C) for 48 h, in patients with CVID ({blacksquare}) and healthy controls ({square}) after addition of different concentrations of the cAMP antagonist Rp-8-Br-cAMPS. Data are given as mean values and SEM. (*), p < 0.1; *, p < 0.05; **, p < 0.01; and ***, p < 0.001. Please note varied scaling of the x-axes.

 
Effects of the cAMP analogs are not specific for IL-10

To determine whether the deficient cytokine secretion in CVID was specific for IL-10 or also affected other cytokines, we measured the secreted levels of IFN-{gamma} and IL-4 in anti-CD3/anti-CD28-stimulated cells from six CVID patients and six healthy controls. These experiments showed reduced levels of both IFN-{gamma} and IL-4 in T cells from CVID comparing healthy controls, but in contrast to IL-10, the differences did not reach statistical significance. Thus, although not specific for IL-10, the impaired anti-CD3/anti-CD28 response in CVID seems to be particularly pronounced for IL-10. As for the effects of the cAMP analogs, we found that the cAMP agonist 8-CPT-cAMP strongly reduced the secreted levels of IFN-{gamma} and IL-4 in anti-CD3/anti-CD28-stimulated T cells from both patients and controls. The cAMP antagonist Rp-8-Br-cAMPS strongly enhanced the secreted levels of IL-4 in both CVID patients (fold increase 4.7 ± 0.74, p = 0.03) and controls (fold increase 3.1 ± 1.08, p = 0.03), but no significant effect was found on IFN-{gamma} in either patients nor controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the hallmark of CVID is defective Ig production in B cells, a substantial proportion of the CVID patients also seems to have a T cell deficiency (6), possibly causing impaired T cell help for B cells (30). In the present study we demonstrate a reduced secretion of IL-10 from T cells in CVID that could not be attributed to a skewed distribution of T cell subsets. Considering the important role of IL-10 in B cell growth and differentiation (10), and the observation that addition of IL-10 to in vitro cultures of B cells from CVID may correct the Ig secretion (14, 15, 16), it is conceivable that reduced IL-10 secretion by T cells may contribute to the deficient B cell function in CVID. Moreover, the reduced secretion of IL-10 seems to involve the cAMP/PKAI system, further suggesting that this system may be of importance in the pathophysiology of CVID as we have previously suggested (20).

There are a some previous studies of IL-10 secretion in CVID reporting enhanced IL-10 secretion from monocytes and secretion comparable to healthy controls in PBMC and purified T cell cultures (31, 32). In these studies, however, supernatants were harvested after 16–24 h. As we show in this study, these time points may not be optimal when studying the ability of T cells to secrete IL-10, as the secreted levels are considerably higher after 48 h. Furthermore, while there are reports that costimulation through the CD28 signaling pathway may correct T cell deficiencies in CVID (33), we found markedly impaired IL-10 release also in T cells costimulated with anti-CD3/anti-CD28.

We and others have previously related raised levels of proinflammatory cytokines such as TNF-{alpha} to a certain phenotypic subgroup of CVID, characterized by splenomegaly, autoimmune disorders, and granulomatous manifestations (34, 35). However, in this study, we found no association between secreted levels of IL-10 in vitro and these clinical and immunological features.

There are several studies of the effect of cAMP-elevating substances on IL-10 production in leukocyte subpopulations, mostly reporting enhancing effects on IL-10 secretion, and this may seem to be in conflict with the present study (21, 22, 23, 24, 25, 36, 37). However, previous studies have often been performed on mixed cell populations that contain monocytes, such as PBMC (21, 24, 36). Thus, while cAMP/PKAI activation does seem to enhance IL-10 synthesis in monocytes, we could demonstrate that such activation markedly suppresses anti-CD3 and anti-CD3/anti-CD28-stimulated IL-10 secretion from purified T cells. This suggests that the effects of cAMP/PKAI activation on IL-10 secretion are different in T cells and monocytes. Moreover, while some authors have found increased IL-10 secretion from T cells using PGE2 as a cAMP-elevating agent (23), possibly also leading to activation of non-cAMP/PKAI-related pathways (38, 39), we demonstrate a suppressive effect of cAMP/PKAI activation on IL-10 levels assessed by a selective PKAI agonist. Finally, as previously demonstrated for T cell proliferation (19), we found that cAMP/PKAI activation did not inhibit the IL-10 secretion following direct protein kinase C (PKC) activation by PMA/calcium ionophore. It has been demonstrated that PKAI is an early inhibitor of T cell activation, acting on the lipid raft-associated pool of C-terminal Src kinase (40) upstream of the effect of PMA and calcium ionophore on PKC. Our findings are in accordance with this notion.

There have been several reports indicating an early defect in the T cell activation in CVID after triggering of the TCR/CD3 complex before the generation of inositol 1,4,5-triphosphate and PKC (20, 41, 42, 43, 44). Specifically, our group has previously demonstrated elevated levels of cAMP in T cells from CVID patients, and that the cAMP antagonist Rp-8-Br-cAMPS may correct the proliferation defect in a subset of these patients (20). In accordance with this, in this study we found that the sensitivity for cAMP-dependent inhibition of IL-10 secretion from anti-CD3/anti-CD28-stimulated T cells was increased in CVID, suggesting a positive cooperative effect of increased endogenous cAMP levels in these patients. Furthermore, we found that a selective inhibition of PKAI markedly improved IL-10 secretion from both anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in CVID. In fact, even at the lowest concentrations of the cAMP antagonist, IL-10 secretion in anti-CD3-stimulated T cells from CVID patients reached levels comparable to those in anti-CD3-stimulated cells from healthy controls. These observations further indicate the involvement of increased cAMP/PKAI activation in the pathogenesis of T cell deficiency in a subset of patients with CVID, and the findings showing normal IL-10 secretion from PMA/calcium ionophore-stimulated T cells in these patients further support such a notion.

In this study, we demonstrate markedly decreased IL-10 secretion in T cells from CVID. However, caution is needed when interpreting the in vivo relevance of this in vitro observation. Thus, during immunological responses several other cells and in particular monocytes/macrophages, may secrete substantial amounts of IL-10. In fact, we have recently found increased rather than decreased serum levels of IL-10 in CVID patients (A. M. Holm, P. Aukrust, S. S. Frøland, unpublished observations), possibly reflecting enhanced release from persistently activated monocytes/macrophages (34, 45). However, close T-B cell interaction is of major importance for B cell activation. Despite the elevated levels of circulating IL-10 that may be present in some patients with CVID, several reports have shown that IL-10, when given concomitantly with anti-CD40, may promote IgG production in B cells from CVID patients in vitro (14, 15, 16). Thus, it is possible that in the microenviroment of T-B cell interaction, IL-10 released from activated T cells acting on B cells in a paracrine manner is more relevant for B cell activation than the circulating IL-10 secreted from monocytes/macrophages.

Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The in vivo significance of this finding should be examined in future studies. Furthermore, our observations suggest the involvement of the cAMP/PKAI system in this defective IL-10 secretion, possibly representing a novel target for therapeutic immunomodulation in future studies of CVID.


   Acknowledgments
 
We thank Bodil Lunden for excellent technical assistance.


   Footnotes
 
1 This work was supported by the Norwegian Research Council. Back

2 Address correspondence and reprint requests to Dr. Are Martin Holm, Research Institute for Internal Medicine, National Hospital, N-0027 Oslo, Norway. E-mail address: a.m.holm{at}klinmed.uio.no Back

3 Abbreviations used in this paper: CVID, common variable immunodeficiency; PKA, protein kinase A; PKAI, PKA type I; 8-CPT-cAMP, 8-(4-chlorophentylthio)cAMP; RP-8-Br-cAMPS, Rp-8-bromo-cAMP-phosphorothioate allophycocyanin; PKC, protein kinase C. Back

Received for publication September 17, 2002. Accepted for publication March 12, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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