Immunological alterations in newly diagnosed primary Sjögren's syndrome characterized by skewed peripheral T-cell subsets and inflammatory cytokines.

OBJECTIVES: To describe how certain peripheral immune parameters reflect the inflammatory alterations in patients with primary Sjögren's syndrome (pSS). We determined lymphocyte subpopulations and their state of activation from peripheral blood, evaluating both soluble serum T-helper (Th)1/Th2-type cytokines and intracytoplasmic cytokines. METHODS: Forty-nine patients with newly diagnosed pSS and 40 healthy individuals, all free from immunomodulant or immunosuppressive medication, were studied. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed using enzyme-linked immunosorbent assay (ELISA), and intracellular cytokine levels were measured after phorbol myristate acetate (PMA) stimulation by flow cytometry after staining of intracellular cytokines. RESULTS: Patients with primary SS had higher percentages of activated CD3+/CD69+ T cells than controls. When comparing naïve vs. memory subsets of CD4+ and CD8+ T cells, a shift towards the memory phenotype was observed for both. Natural killer (NK) cell and NK T-cell (NKT) percentages and Th0 and Th1 cell numbers were increased in patients compared to controls. Among circulating cytokines, interferon (IFN)-gamma was high, whereas interleukin (IL)-10 was decreased in SS when compared to controls. CONCLUSIONS: SS, considered as a systemic autoimmune disease, is characterized by a complex interplay of various cytokines and immune cells. The skewed T-cell subsets and cytokine imbalance play important roles in an orchestrated proinflammatory cascade.

The severity of systemic lupus erythematosus negatively correlates with the increasing number of CD4+CD25(high)FoxP3+ regulatory T cells during repeated plasmapheresis treatments of patients.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by increased pathologic autoantibody production. A decrease in the number of CD4+CD25(high)FoxP3+ regulatory T cells can play a key role in the loss of tolerance to self antigens. Our aim was to determine the absolute number of peripheral CD4+CD25(high)FoxP3+ T cells in 44 patients with SLE, furthermore, to measure the changes in the number of CD+CD25(high)FoxP3+ T cells in 5 patients with severe SLE treated with repeated plasmapheresis for 4-6 days in comparison to the changes in the activity of disease (SLEDAI). Percent of CD4+CD25(high)FoxP3+ T cells were measured by flow cytometry. The absolute number of peripheral CD4+CD25(high)FoxP3+ T cells was significantly decreased in the 44 patients with SLE compared to the healthy controls n = 32 (0.012 +/- 0.006 vs. 0.038 +/- 0.017 G/L, p < 0.05). In the 5 patients with severe SLE the repeated plasmapheresis treatments increased the peripheral number of CD4+CD25(high)FoxP3+ T cells. As the number of CD4+CD25(high)FoxP3+ T cells increased during the treatment, the activity of disease (the value of SLE activity index) decreased. In the peripheral blood of SLE patients not only the ratio was decreased (as it was published earlier) but also the absolute number of these regulatory T cells. The repeated plasmapheresis treatments of SLE patients induced a significant increase in the number of peripheral CD4+CD25(high)FoxP3+ T cells in parallel to the decrease in the values of SLEDAI (the activity of disease). This phenomenon is, among others, possibly due to the elimination of interpheron-alpha and lymphocytotoxic antibodies during plasmapheresis.

Measurement of natural (CD4+CD25high) and inducible (CD4+IL-10+) regulatory T cells in patients with systemic lupus erythematosus.

Abnormalities of regulatory T cells may play an important role in the loss of self-tolerance, which is a major characteristic of lupus. The objective of this study was to determine the ratio and the number of natural CD4+CD25highFoxp3+ and inducible CD4+IL-10+ regulatory T cells in lupus patients and to search correlation with disease activity. Seventy-two Hungarian lupus patients were enrolled in the study. Fourty-one age- and sex matched healthy donors served as controls. Flow cytometry was used for the quantification of CD4+CD25high Foxp3+ (nTreg) and CD4+IL-10+ (iTreg) cells. The ratio (3.06 +/- 1.45%) and the number (0.019 +/- 0.012 x 10(9)/L) of nTreg cells decreased in lupus significantly (P < 0.001 in both) as compared to normal controls (4.26 +/- 1.01% and 0.039 +/- 0.017 x 10(9)/L). The ratio of iTreg cells were significantly higher in patients than in controls (20.92 +/- 14.02% versus 15.49 +/- 11.65%, P < 0.03), but the number of these cell type did not differ in significant manner (0.314 +/- 0.236 x 10(9)/L versus 0.259 +/- 0.183 x 10(9)/L). The 19 active patients were characterised by significantly higher disease activity index (SLEDAI 8.63 +/- 2.95 versus 1.74 +/- 1.68, P < 0.001) and anti-DNA concentration (117.85 +/- 145.89 versus 37.36 +/- 68.85 IU/mL, P = 0.001) as compered to the 52 inactive patients. Furthermore, active patients required higher dose of methylprednisolon than inactive ones (14.8 +/- 10.6 versus 4.8 +/- 3.4 mg/day, P < 0.001). However, we did not find statistical significant difference in the number and ratio of the examined cell populations regarding to disease activity. Altered ratio and number of both natural and inducible regulatory T cells may play a role in the pathogenesis of lupus. There are small but appreciable difference in the number of regulatory T cells between inactive patients and healthy controls. It suggests that immunoregulatory deficiencies are present in the inactive stage of the disease also.

TH1/TH2 imbalance, measured by circulating and intracytoplasmic inflammatory cytokines--immunological alterations in acute coronary syndrome and stable coronary artery disease.

To describe how peripheral immune-parameters reflect the inflammatory alterations of the atherosclerotic plaques in coronary atherosclerosis. We measured general inflammatory markers C-reactive protein (CRP) and granulocyte activity, lymphocyte subpopulations and their state of activation, evaluated circulating Th1/Th2-type cytokines, and specific intracytoplasmic cytokines. We investigated the association of immune-parameters with disease outcome and mortality. Thirty-three patients with acute coronary syndrome (ACS), 62 with stable coronary artery disease (CAD) and 58 healthy controls were studied. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines and autoantibodies were assessed using enzyme-linked immunosorbent assay (ELISA), while intracellular cytokine levels were measured by flow cytometry after intracellular staining. We found elevated levels of CRP and granulocyte activity in ACS versus CAD (P < 0.001, P = 0.017, respectively). Natural killer (NK) cell percentages were elevated, while percentage of T cells to the total lymphocyte count was slightly decreased in ACS compared to controls (P < 0.0001, P = 0.012, respectively). Both forms of coronary atherosclerosis showed significantly higher percentages of activated T cells than controls when stained for the activation markers HLA-DR3 and CD69(+) (ACS: P < 0.0001, P = 0.002, CAD: P < 0.0001, P = 0.018, respectively). IL-1, IL-4 and IL-10 proved significantly higher in ACS versus controls (P = 0.036, P = 0.01, P < 0.0001 respectively). Th1 to Th2 ratio shifted towards a Th1 dominance in both diseases. Both general proinflammatory markers and activated T cells signify CAD. The orchestrated proinflammatory cascade eventually leads to the development of the disease.

Regulatory T cells in peripheral blood of patients with mixed connective tissue disease.

OBJECTIVE: To determine the percentage and absolute number of CD4+ regulatory T-cells (Treg) in 48 patients with mixed connective tissue disease (MCTD). METHODS: Data were classified on the basis of the stage of the disease: 17 patients were in the active stage and 31 in the inactive stage. The absolute number of CD4+/intracellular interleukin-10+ (IL-10+) and CD4+CD25+high Treg cells was determined by flow cytometry. The percentage of CD4+CD25+high suppressor T-cells was determined on the basis of Foxp3 expression. RESULTS: The percentage and the absolute number of CD4+CD25+high Treg cells were lower in patients than in healthy controls (p<0.04), and were further decreased in patients with active MCTD and were lower than in the inactive stage (p<0.01). There was an increase in the percentage and absolute number of CD4+IL-10+ Treg cells in patients with MCTD compared to the healthy controls (p<0.02). The percentage of CD4+IL-10+ Treg cells was higher in the active stage of MCTD than in the inactive stage of the disease (p<0.005). However, we did not find any significant difference in the absolute number of CD4+IL-10+ Treg cells between the patients in the active and inactive stages of MCTD. CONCLUSION: Our results suggest that the decrease in the number of CD4+CD25+high Treg cells in an important factor in the immunoregulatory disturbance in patients with MCTD. We suggest that the increase in the number of CD4+IL-10+ Treg cells is a compensatory mechanism aiming to restore the balance between type 1 and type 2 cytokines in MCTD.

Clinical and immunoserological characteristics of mixed connective tissue disease associated with pulmonary arterial hypertension.

We investigated the clinical characteristics and immunoserological alterations in patients with mixed connective tissue disease (MCTD) associated with pulmonary arterial hypertension (PAH). Anti-U1RNP autoantibodies, anti-endothelial cell antibodies (AECA) and serum thrombomodulin (TM) as well as von Willebrand factor antigen (vWFAg) concentrations were measured in 25 patients with MCTD associated with PAH and in 154 MCTD patients without PAH. The results showed that the probability of survival was lower in MCTD patients with PAH than in the 154 MCTD-non-PAH patients (5-year survival rate in MCTD with PAH: 73%, versus 96% in MCTD-non-PAH; P < 0.01). AECA were more frequently present in the sera of MCTD patients with PAH than in MCTD-non-PAH (P < 0.001). Serum TM and vWFAg levels were higher in MCTD-PAH patients than in MCTD-non-PAH patients (TM: P < 0.001; vWFAg: P < 0.001). Significant correlation was noticed between the quantity of AECA and TM level (r = 0.466) as well as the quantity of AECA and vWFAg level (r = 0.550). In conclusion, our results suggest that in MCTD the presence of AECA and endothelial cell activation may play a role in the development of PAH and in the maintenance of obliterative vascular processes.

Altered cytokine expression of peripheral blood lymphocytes in polymyositis and dermatomyositis.

OBJECTIVE: To investigate the intracellular and soluble cytokine levels and T cell subsets in peripheral blood of patients with active and inactive polymyositis and dermatomyositis. METHODS: The frequencies of T and B lymphocytes, T helper (Th), and T cytotoxic (Tc) cells and of interferon gamma (IFNgamma), interleukin (IL)4, and IL10 expression of CD4+ or CD8+ cells were determined by flow cytometry. The concentrations of soluble cytokines were measured with commercial enzyme linked immunosorbent assays. RESULTS: In active dermatomyositis there was a decreased percentage of T (CD3+) lymphocytes and Tc (CD8+) lymphocytes, decreased IFNgamma expression of CD4+ and CD8+ cells, but an increase in B and IL4 producing CD4+ lymphocyte frequencies. These prominent changes disappeared in the inactive stage of the disease. In polymyositis no significant change in these lymphocyte subsets or in intracellular cytokine expression could be detected in either the active or the inactive form. The frequency of IL4+/IFNgamma+ Th cells was calculated and a significantly increased Th2/Th1 frequency was found in active dermatomyositis, and a decreased frequency in inactive dermatomyositis, compared with the control population. CONCLUSIONS: There appears to be a difference between polymyositis and dermatomyositis in the level of peripheral blood lymphocytes and their intracellular cytokine content. These findings provide further evidence for a difference in the pathogenesis of polymyositis and dermatomyositis.

Ultraviolet-A1 phototherapy modulates Th1/Th2 and Tc1/Tc2 balance in patients with systemic lupus erythematosus.

OBJECTIVE: Ultraviolet-A1 (UVA1) phototherapy is effective for a variety of dermatological diseases. We examined the effectiveness and reliability of low-dose UVA1 phototherapy (60 kJ/m2/treatment) in patients suffering from systemic lupus erythematosus (SLE). We studied the changes in immunological parameters. METHODS: The patients received a 9-week course of phototherapy according to the following regimen: five times a week during the first 3 weeks, three times a week during the second 3 weeks and twice during the last 3 weeks. Among other things, we analysed the proportions of T helper 1 (Th1), Th2, T cytotoxic (Tc1) and Tc2 cell populations in the peripheral blood of patients by flow cytometric detection of intracytoplasmic interferon gamma (IFN-gamma) and interleukin 4 (IL-4). RESULTS: Our study showed the improvement of clinical symptoms determined by the subjective clinical disease activity scoring and the SLE Disease Activity Index (SLEDAI). By the end of UVA1 phototherapy, the mean value of SLEDAI had decreased from 7.2+/-5.6 to 0.9+/-1.8, which was significant (P = 0.005). Immunological investigations detected a decrease in the frequency of IFN-gamma-producing Th1 and Tc1 cells and a decrease in the Th1/Th2 and Tc1/Tc2 ratios after UVA1 therapy. CONCLUSION: According to the literature, IFN-gamma has a pathogenic role in the development of SLE. We observed a decreased proportion of IFN-gamma-secreting cells, which we think is presumably one of the beneficial effects of UVA1 therapy. On the basis of our study, UVA1 phototherapy does seem to be an effective adjuvant in the treatment of SLE patients.

Abnormal cell-specific expressions of certain protein kinase C isoenzymes in peripheral mononuclear cells of patients with systemic lupus erythematosus: effect of corticosteroid application.

We have studied the expressions of various protein kinase C (PKC) isoenzymes in T cells and monocytes from patients with systemic lupus erythematosus (SLE), in comparison to those of healthy controls and patients with other immunological disorders. As measured by Western blotting, the levels of PKCbeta, delta, eta, epsilon, theta and zeta (but not of PKCalpha) significantly decreased in T cells of SLE patients. In monocytes, however, we observed marked suppressions only in the expressions of PKCdelta, epsilon and zeta but not in the expressions of other PKC isoforms. In vivo corticosteroid application, as well as in vitro steroid treatment of monocytes, elevated the expressions of most isoforms close to normal values; however, the decreased levels of PKCtheta and zeta were not affected by steroid application. These alterations were characteristic to SLE because we could not detect any changes in the PKC levels in mononuclear cells of primary Sjögren's syndrome and mixed connective tissue disease patients. These results suggest that impaired PKC isoenzyme pattern may exist in the T cells and monocytes of SLE patients. Furthermore, the clinically efficient glucocorticoid application in SLE can increase the expression of some members of PKC system.

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes.

We have previously shown that the protein kinase C (PKC) system plays a pivotal role in regulation of proliferation and differentiation of the human keratinocyte line HaCaT which is often used to assess processes of immortalization, transformation, and tumorigenesis in human skin. In this paper, using pharmacological and molecular biology approaches, we investigated the isoform-specific roles of certain PKC isoenzymes (conventional cPKCalpha and beta; novel nPKCdelta and epsilon) in the regulation of various keratinocyte functions. cPKCalpha and nPKCdelta stimulated cellular differentiation and increased susceptibility of cells to actions of inducers of apoptosis, and they markedly inhibited cellular proliferation and tumor growth in immunodeficient mice. In marked contrast, cPKCbeta and nPKCepsilon increased both in vitro and in vivo growth of cells and inhibited differentiation and apoptosis. Our data present clear evidence for the specific, antagonistic roles of certain cPKC and nPKC isoforms in regulating the above processes in human HaCaT keratinocytes.

Association between the occurrence of the anticardiolipin IgM and mite allergen-specific IgE antibodies in children with extrinsic type of atopic eczema/dermatitis syndrome.

BACKGROUND: As the literature has only controversial data on the role of nonallergen-specific antibodies in atopic eczema dermatitis syndrome, the authors investigated the link between the occurrence of the antiphospholipid [anticardiolipin (ACL), anti-beta2-glycoprotein I] and allergen-specific immunoglobulin E (IgE) antibodies in 72 children with atopic eczema/dermatitis syndrome (AEDS). METHODS: The measurement of antiphospholipid antibodies was carried out by enzyme-linked immunosorbent assay (ELISA), serum total IgE by nephelometry, and allergen-specific IgE by immunoblotting assay. The statistical analysis was carried out by Fisher's exact test and odds ratio was calculated. RESULTS: Thirteen of 72 children with AEDS (mean age 8.3 years) had elevated serum levels of ACL, and eight anti-beta2-glycoprotein I antibodies. The presence of allergen-specific IgE against inhalant allergens and nutritive allergens was among eight of 13 and three of eight in the cases with elevated ACL. The ratio of patients with highly increased severity scoring of atopic dermatitis (SCORAD) index (>75) was significantly higher in the group with elevated (4/13) than in those with the normal ACL levels (2/59). There was a significant association between the appearance of mite (Dermatophagoides pteronyssinus, D. farinae)-specific IgE and ACL IgM antibodies (6/13). CONCLUSION: These findings show that there are significant linkage and association between the appearance of ACL IgM or the production of allergen-specific IgE against inhalant (mainly mite) allergens in children with atopic eczema/dermatitis syndrome.

Increased frequency of intracellular interleukin (IL)-13 and IL-10, but not IL-4, expressing CD4+ and CD8+ peripheral T cells of patients with atopic dermatitis.

BACKGROUND: A number of studies exist demonstrating the increased expression of type 2 cytokines and decreased capacity to produce interferon-gamma (IFN-gamma) in peripheral blood mononuclear cells (PBMCs) of patients with atopic dermatitis (AD). OBJECTIVES: To clarify the results of recent studies concerning the role of interleukin (IL)-4 and IL-13 in PBMCs of AD patients, we analysed the activation status of lymphocyte subpopulations. METHODS: We measured the intracellular expression and serum levels of certain type 1 and type 2 cytokines, using cell surface and intracellular cytokine staining, flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS: The frequency of IL-10 and IL-13 producing CD4+ and CD8+ T cells was significantly higher in patients with AD, while the frequency of IFN-gamma secreting helper and cytotoxic T cells was significantly lower in patients with AD than in control subjects. The serum levels of IL-10 and IL-13 were also significantly increased. There were no significant differences observed between the experimental groups in the frequency of IL-4 producing CD4+ and CD8+ cells. CONCLUSIONS: This study demonstrates a type 2 cytokine production in the CD4+ and CD8+ T cells of AD patients, which is characterized by an elevated IL-13, but not by IL-4 secretion, and by an increased level of the immunoregulatory IL-10, which can contribute to a decrease in IFN-gamma expression.

Decreased arachidonic acid release in peripheral blood monocytes of patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the release of arachidonic acid (AA) in unfractionated peripheral blood mononuclear cells (PBMC), separated monocytes and T lymphocytes of patients with systemic lupus erythematosus (SLE). METHODS: AA release was measured in cells from 56 patients with SLE and from 48 controls. Of the 56 patients with SLE, 38 were receiving glucocorticosteroids and 18 were not. [3H]AA was incorporated into the membranes of PBMC and purified subsets of monocytes and T lymphocytes. The release of [3H]AA was measured both in nonstimulated cells cultured for 24 h and in cell cultures stimulated by phorbol ester (PMA) and Ca2+ ionophore for 4 h. RESULTS: In the PBMC of SLE patients not taking glucocorticosteroids, the release of AA was decreased in both stimulated and nonstimulated cells. There was a decrease of AA production in monocytes but not in T lymphocytes. This phenomenon could be observed in the active and inactive phases of the disease. CONCLUSION: A defect in AA production may exist in the peripheral monocytes of patients with SLE, resulting in decreased release of AA in patients not receiving glucocorticosteroid therapy.

Measurement of intracellular interferon-gamma and interleukin-4 in whole blood T lymphocytes from patients with systemic lupus erythematosus.

Contradictory data are available about the dominance of T-helper 1 (T(H)1), or T-helper 2 (T(H)2) cytokines in systemic lupus erythematosus (SLE). Therefore, intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production of T lymphocytes was measured in whole blood of healthy donors and active and inactive SLE patients by flow cytometry. The percentage of IFN-gamma and IL-4 positive cells was low (<1%) in unstimulated samples of the healthy controls, while that of IFN-gamma and IL-4 positive cells in the stimulated cells was 25.2+/-10.6% and 0.6+/-1.5%, respectively. No significant difference was found between SLE patients and healthy controls and between active and inactive patients in these parameters either in the unstimulated or in the stimulated samples. One patient with severe disease had as high as 11.8% IL-4 positive cells and 12.5% IFN-gamma positive cells in the stimulated samples, but after the initiation of intensive corticosteroid and cytostatic therapy, the percentage of IL-4 positive T cells decreased (4.76%) while that of IFN-gamma positive T cells increased (47.91%). We conclude that the intracellular IL-4 and IFN-gamma expression of T lymphocytes does not differ markedly between SLE patients and healthy controls, with the possible exception of severe disease, when marked IL-4 overproduction may exist beside low IFN-gamma production. Furthermore, corticosteroid and cytostatic therapy might normalize this altered IFN-gamma/IL-4 ratio.