Epstein-Barr virus induces normal B cell responses but defective suppressor T cell responses in patients with systemic lupus erythematosus

Epstein-Barr virus (EBV) is a polyclonal B cell activator, independent of helper T cells, which induces the generation of suppressor T cells in vivo and in vitro. Given the complexity of the immunologic abnormalities in patients with systemic lupus erythematosus (SLE), we used EBV as a tool to examine the following questions: a). Are SLE B cells primarily defective? and b). Does EBV stimulate the generation of suppressor activity from SLE T cells? It was found that B cells from SLE patients infected with EBV in vitro generate plaque-forming cell (PFC) responses that are similar to those raised by normal B cells infected with EBV within the first 14 days of culture. T cells from SLE patients, in contrast to T cells from normal individuals, cultured with autologous B cells plus EBV fail to develop the expected normal decrement of PFC during the late phase of the in vitro culture (day 14). However, B cells from SLE patients are susceptible to suppression as mixed cultures of SLE B cells and normal allogeneic T cells showed a pattern of PFC response to EBV similar to that of the co-culture of normal B cells with normal T cells. T cells from SLE patients, in analogous mixed cell cultures, failed to suppress either normal B cells or allogeneic SLE B cells. The above experiments indicate that the B cells are not intrinsically defective in SLE patients; rather, a specific T cell abnormality contributes to the lack of normal immunoregulation of certain B cell responses in SLE.     

Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19(+)/CD27(-) naive B cells more than CD19(+)/CD27(+) memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27(high)/CD38(+)/CD19(dim)/surface Ig(low)/CD20(-)/CD138(+) plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27(high) plasma cells and naive CD27(-) B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27(+) B cells coexpressing IgD were memory B cells preferentially using V(H)3 family members with multiple somatic mutations. CD27(high) plasma cells showed a similar degree of somatic hypermutation, but preferentially employed V(H)4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.     

Soluble granzyme B and cytotoxic T lymphocyte activity in the pathogenesis of systemic lupus erythematosus

Activated cytotoxic T lymphocyte (CTL) mediated target cell death has been implicated in the development of systemic autoimmune disease like SLE. However, the role of soluble granzyme B and its relationship with CTL activity and disease activity is still unknown. In this study, we evaluated role of soluble granzyme B and cytotoxic T lymphocyte activity in SLE patients. The soluble granzyme B was measured in the serum by an enzyme-linked immunosorbent assay while cytotoxic T lymphocyte activity was measured by flow cytometry. The disease activity was determined by using SLE Disease Activity Index (SLEDAI) score. Cytotoxic T lymphocyte activity was increased and strongly associated with disease activity. The soluble granzyme B levels were higher in SLE patients and associated with various clinical features like reduced complement components; C3 & C4 and skin lesion. The soluble granzyme B levels were also sturdily related with severity of the disease. The findings of this study suggest that excessive secretion of soluble granzyme B and enhanced activity of cytotoxic T lymphocyte may play a vital role in the pathogenesis of SLE and organ damage. Also, evaluation of soluble granzyme B may be helpful in monitoring the clinical features associated with activated CTL in SLE.     

Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus

Alteration of T cell suppression function has been recognized in patients with systemic lupus erythematosus (SLE). Recently, CD8(+) T suppressor lymphocytes (CD8(+) Ts) have been generated in vitro by incubating purified CD8(+) T cells with IL-2 and GM-CSF. Using this method, we generated CD8(+) Ts from patients affected by SLE. No major differences were found in the CD8(+) Ts phenotype between SLE patients and healthy subjects. CD8(+) Ts from SLE patients with active disease did not inhibit the anti-CD3 mAb-induced proliferation of autologous PBMC, whereas CD8(+) Ts from SLE patients in remission exerted an inhibitory activity comparable to normal subjects. The inhibitory effect of CD8(+) Ts cells was neither mediated by cytotoxic activity nor by apoptosis induction. Two cytokines, IFN-gamma and IL-6, were found to be responsible for the function of CD8(+) TS: In fact, counteraction of CD8(+) Ts suppression activity was obtained by blocking IFN-gamma with a specific Ab or by inhibiting CD8(+) Ts-mediated IL-6 secretion by an antisense oligonucleotide. Interestingly, CD8(+) Ts from SLE patients showed a peculiar cytokine pattern characterized by an impaired secretion of IL-6 and an increased secretion of IL-12. Thus, it appears that an altered balance between inhibitory (IL-6) and stimulatory (IL-12) cytokines might be responsible for the functional impairment of CD8(+) Ts in SLE patients.     

Characterization of the human T lymphocyte adenosine receptor: comparison of normal and systemic lupus erythematosus cells

To determine whether a defect in the T cell response to adenosine exists at the level of the adenosine receptor in systemic lupus erythematosus (SLE) we measured the binding affinity and maximum binding of T cell membranes from both normal and SLE T cells by utilizing radiolabeled adenosine ligands. Normal T lymphocyte membranes possess a single class of [3H]5-N-ethylcarboxamide adenosine binding sites with a Kd of 0.61 microM, a Bmax of 23.5 pmol/mg protein, and a Hill coefficient of 0.98, which indicates the presence of noncooperative sites. In contrast, T cell membranes do not bind significant amounts of either [3H]cyclohexyladenosine or [3H]phenylisopropyladenosine. These data indicate that T lymphocyte membranes have only A2, and not A1, adenosine receptors. Similarly, T cells from both active and inactive SLE subjects also express only A2 receptors with a Kd of 0.93 microM, a Bmax of 20.4 pmol/mg protein, and a Hill coefficient of 0.85, which is consistent with the presence of noncooperative sites. There is no difference in the on-rate, affinity, or density of T cell A2 receptors from active SLE patients, inactive SLE patients, or healthy controls. We conclude that T lymphocytes from both healthy and SLE subjects express A2, but not A1, receptors. Thus, the inability of SLE T cells to respond to adenosine does not reflect a decreased density of A2 (stimulatory) receptors, diminished A2 receptor binding, or an increased affinity or number of A1 (inhibitory) adenosine receptors. These observations support the conclusion that the defect in the T cell cAMP-dependent pathway may occur at a point distal to the adenosine receptor.     

Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.     

Association between T lymphocyte sub-sets apoptosis and peripheral blood mononuclear cells oxidative stress in systemic lupus erythematosus

Increased oxidative stress and lymphocyte apoptosis are a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). However, the association between oxidative stress and T lymphocytes apoptosis has still to be elucidated in SLE. In order to appraise the interaction between oxidative stress and T lymphocyte apoptosis with the severity of disease, oxidative stress profile and T lymphocytes apoptosis were studied. Increased levels of ROS, MDA and CD4(+) lymphocyte apoptosis were positively associated with disease activity while decreased levels of GSH and percentage expression of CD4(+) lymphocyte were negatively associated with disease activity. The decrease in intracellular levels of GSH was negatively associated with T lymphocyte, CD4(+) lymphocyte, CD8(+) lymphocyte apoptosis and intracellular caspase-3 expression. The present study suggests that increased T lymphocyte sub-sets apoptosis may be mediated by decreased intracellular glutathione concentration and severity of disease might be enhanced together by over-production of ROS in SLE.     

Characterization of T lymphocyte subpopulations responsible for deficient interleukin 2 activity in patients with systemic lupus erythematosus

Normal immunoregulation depends on a complex set of cellular interactions in which interleukin 2 (IL 2) appears to play an important role. We have examined the IL 2 activity in patients with systemic lupus erythematosus (SLE). IL 2 production by phytohemagglutinin (PHA)-stimulated T cells for 48 hr was measured by the ability of their culture fluid to induce proliferation of normal human T cells that had been activated for more than 20 days by PHA plus IL 2. To measure IL 2 responsiveness, T cells were blasted by preincubation with concanavalin A for 96 hr and stimulated for another 72 hr with lectin-free standard IL 2. SLE T cells failed to produce normal levels of IL 2 in vitro compared with normal control T cells. This failure resided in both OKT4+ and OKT8+ cells. Furthermore, the abnormality was due neither to soluble inhibitory factors produced by SLE T cells nor to active suppressor cells that might be induced by PHA-stimulation. Responsiveness to IL 2 of T cells from some, but not all, SLE patients was decreased significantly from that of normal controls. Absorption studies as well as studies with anti-Tac antibody demonstrated that the impaired responsiveness of T cells in the specific patients with SLE was due to inadequate expression of IL 2 receptors on the T cells upon activation. This defect was exclusively ascribed to the dysfunction of OKT4+, but not OKT8+, cells. The above defects in production of and responsiveness to IL 2 observed in patients with SLE were present at all times regardless of the disease activity or of corticosteroid therapy. Thus, the deficient IL 2 activity may be intrinsic to SLE lymphocytes and may contribute to impaired immunoregulation and to the development of SLE.     

Association between T lymphocyte sub-sets apoptosis and peripheral blood mononuclear cells oxidative stress in systemic lupus erythematosus

Abstract

Increased oxidative stress and lymphocyte apoptosis are a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). However, the association between oxidative stress and T lymphocytes apoptosis has still to be elucidated in SLE. In order to appraise the interaction between oxidative stress and T lymphocyte apoptosis with the severity of disease, oxidative stress profile and T lymphocytes apoptosis were studied. Increased levels of ROS, MDA and CD4⁺ lymphocyte apoptosis were positively associated with disease activity while decreased levels of GSH and percentage expression of CD4⁺ lymphocyte were negatively associated with disease activity. The decrease in intracellular levels of GSH was negatively associated with T lymphocyte, CD4⁺ lymphocyte, CD8⁺ lymphocyte apoptosis and intracellular caspase-3 expression. The present study suggests that increased T lymphocyte sub-sets apoptosis may be mediated by decreased intracellular glutathione concentration and severity of disease might be enhanced together by over-production of ROS in SLE.     

B cell and T cell lymphopenia in systemic lupus erythematosus

The absolute number and proportion of B and T lymphocytes in the peripheral blood was determined in 16 active and 11 inactive patients with systemic lupus erythematosus. The total number of B and T lymphocytes was significantly reduced when compared to normals. The population of thymus-derived cells seems to be the one predominately affected. These abnormalities improve, but are not completely reversed, when patients are in remission.     

Deficiencies in suppressor T cell activity seen in patients with active systemic lupus erythematosus are due to the dilution of normally functioning suppressor T cells by nonsuppressor T cells

Concanavalin A (Con A)-activated T lymphocytes from patients with active, but not inactive, systemic lupus erythematosus (SLE) failed to express normal suppressor activity, regardless of the phenotype of CD4+ or CD8+. Con A-activated CD4+ or CD8+ T lymphocytes from the SLE patients and from normal controls were further separated into two populations, using the autologous erythrocyte rosette technique. One population very rich in cells capable of forming rosettes with autologous erythrocytes from the active patients showed the same degree of suppressor activity, as did that from normal controls; the CD4+ or CD8+ population poor in autorosetting cells derived from Con A-activated T lymphocytes from both the controls and patients did not express suppressor activity. Moreover, when autorosetting T cells from the active patients and nonrosetting cells from the same patients were mixed at a normal ratio (4:6), normal suppressor activity could be restored. It was notable that the frequency of autorosette-forming cells was markedly reduced in the Con A-activated T lymphocytes from the active, but not inactive, SLE patients, regardless of the phenotype of CD4+ or CD8+. These findings indicate the presence of a normally functioning suppressor T cell population in patients with active SLE. It seems that the lack of suppressor T cell function in patients with active SLE is due to the dilution of a few normal suppressor T cells by large numbers of nonsuppressor T lymphocytes.     

Regulatory T cells in systemic lupus erythematosus: past,present and future

Regulatory/suppressor T cells (Tregs) maintain immunologic homeo-stasis and prevent autoimmunity. In this article, past studies and recent studies of Tregs in mouse models for lupus and of human systemic lupus erythematosus are reviewed concentrating on CD4⁺CD25⁺Foxp3⁺ Tregs. These cells consist of thymus-derived, natural Tregs and peripherally induced Tregs that are similar phenotypically and functionally. These Tregs are decreased in young lupus-prone mice, but are present in normal numbers in mice with established disease. In humans, most workers report CD4⁺Tregs are decreased in subjects with active systemic lupus erythematosus, but the cells increase with treatment and clinical improvement. The role of immunogenic and tolerogenic dendritic cells in controlling Tregs is discussed, along with new strategies to normalize Treg function in systemic lupus erythematosus.     

T cell studies in a peptide-induced model of systemic lupus erythematosus

We have previously reported that immunization with a peptide mimetope of dsDNA on a branched polylysine backbone (DWEYSVWLSN-MAP) induces a systemic lupus erythematosus-like syndrome in the nonautoimmune BALB/c mouse strain. To understand the mechanism underlying this breakdown in self tolerance, we examined the role of T cells in the response. Our results show that the anti-foreign and anti-self response induced by immunization is T cell dependent and is mediated by I-E(d)-restricted CD4(+) T cells of the Th1 subset. In addition, generation of the critical T cell epitope requires processing by APCs and depends on the presence of both DWEYSVWLSN and the MAP backbone. The breakdown in self tolerance does not occur through cross-reactivity between the T cell epitope of DWEYSVWLSN-MAP and epitopes derived from nuclear Ags. In this induced-model of SLE, therefore, autoreactivity results from the activation of T cells specific for foreign Ag and of cross-reactive anti-foreign, anti-self B cells. Despite the fact that tissue injury is mediated by Ab, the critical initiating T cell response is Th1.     

Global natural regulatory T cell depletion in active systemic lupus erythematosus

The immune defect that could account for the multisystemic involvement that characterizes systemic lupus erythematosus (SLE) remains unknown. We hypothesized that iterative disease flares correspond to a recurrent defect in the peripheral immune suppression exerted by naturally occurring T regulatory cells (Tregs). Surprisingly, Tregs isolated from lupus patients show the same phenotypic and functional characteristics as corresponding cells found in healthy controls. A decrease in the proportion of circulating Tregs among other CD4+ T cells is nevertheless evidenced in active patients when this group is compared with healthy controls (0.57 +/- 0.24%, n = 45 vs 1.29 +/- 0.38%, n = 82, p < 0.0001) or with inactive patients (1.22 +/- 0.67%, n = 62, p < 0.0001). In contrast, the proportion of Tregs in other systemic autoimmune diseases such as primary Sj?gren syndrome and inflammatory myopathy does not significantly differ from controls' values (1.15 +/- 0.46%, n = 21, p = 0.09 and 1.16 +/- 0.44%, n = 16, p = 0.43, respectively). Lupus Tregs do not accumulate in either the lymph nodes or the diseased kidneys and are not killed by a circulating soluble factor, but demonstrate in vitro a heightened sensitivity to Fas-induced apoptosis. Finally, we show that the extent of Treg depletion correlates with the clinical severity of the flare. SLE flares are therefore associated with a global Treg depletion and not with a phenomenon of tissue redistribution. In summary, we suggest that the physiopathology of SLE could be tied to a defect in the homeostatic control of the Treg subpopulation.     

Deoxyribonuclease-Inhibitory antibodies in systemic lupus erythematosus

Deoxyribonucleases (DNases) are key enzymes for digesting DNA. Abnormalities in the function of these enzymes may contribute to the development of anti-DNA antibodies in systemic lupus erythematosus (SLE). In this study, we used bovine DNase 1-coated ELISA plates to screen anti-DNase antibodies in SLE patients. About 62% of the sera of SLE patients (63/101) were positive for anti-DNase antibodies compared to only 8% of normal controls (8/98). A positive correlation was also found between the concentrations of anti-DNase and anti-DNA antibodies in sera of SLE patients. Affinity-purified anti-DNase immunoglobulin G (IgG) from pooled sera of SLE patients bound to bovine DNase as well as DNA. A synthetic peptide, corresponding to the catalytic site of DNase, was able to completely inhibit the binding of anti-DNase IgG to DNase. In addition to bovine DNase, the anti-DNase IgG also bound to and inhibited the enzymatic activities of DNase present in streptococcal supernatants and human urine. Immunization of lupus-prone NZB/NZW mice with bovine DNase enhanced the production of anti-DNase and DNA antibodies, and accelerated the occurrence of proteinuria. Taken together, these results suggest that DNase-inhibitory antibodies which recognize a conserved epitope near the catalytic site of DNase may act in the pathogenesis of SLE.