Jorge Alcocer-Varela

A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans

Systemic lupus erythematosus (SLE, OMIM 152700) is a complex autoimmune disease that affects 0.05% of the Western population, predominantly women. A number of susceptibility loci for SLE have been suggested in different populations, but the nature of the susceptibility genes and mutations is yet to be identified. We previously reported a susceptibility locus (SLEB2) for Nordic multi-case families. Within this locus, the programmed cell death 1 gene (PDCD1, also called PD-1) was considered the strongest candidate for association with the disease. Here, we analyzed 2,510 individuals, including members of five independent sets of families as well as unrelated individuals affected with SLE, for single-nucleotide polymorphisms (SNPs) that we identified in PDCD1. We show that one intronic SNP in PDCD1 is associated with development of SLE in Europeans (found in 12% of affected individuals versus 5% of controls; P = 0.00001, r.r. (relative risk) = 2.6) and Mexicans (found in 7% of affected individuals versus 2% of controls; P = 0.0009, r.r. = 3.5). The associated allele of this SNP alters a binding site for the runt-related transcription factor 1 (RUNX1, also called AML1) located in an intronic enhancer, suggesting a mechanism through which it can contribute to the development of SLE in humans.

Clinical and biologic effects of anti–interleukin-10 monoclonal antibody administration in systemic lupus erythematosus

OBJECTIVE To evaluate the safety and clinical efficacy of administering an anti-interleukin-10 (anti-IL-10) monoclonal antibody (mAb) to systemic lupus erythematosus (SLE) patients with active and steroid-dependent disease. In addition, we sought to assess the effects of in vivo IL-10 neutralization on biologic markers of SLE. METHODS Treatment consisted of 20 mg/day intravenous administration of an anti-IL-10 murine mAb (B-N10) for 21 consecutive days, with a followup period of 6 months. Six patients were studied. RESULTS Treatment was safe and well tolerated. All patients developed antibodies against B-N10. Cutaneous lesions and joint symptoms improved in all patients beginning during B-N10 administration and continuing to month 6. The SLE Disease Activity Index decreased from a mean +/- SEM of 8.83+/-0.91 on day 1 to 3.67+/-0.67 on day 21 (P = 0.001), 1.50+/-0.84 at month 2, and 1.33+/-0.80 at month 6 (P<0.001). At the end of followup, the disease was clinically inactive in 5 of the 6 patients. Prednisone administration was decreased from a mean +/- SEM of 27.9+/-5.7 mg/day on day 1 to 9.6+/-2.0 mg/day at month 6 (P<0.005). Activity of immune and endothelial cells rapidly decreased, as assessed by the early evolution of several biologic markers. CONCLUSION This is the first report of IL-10 antagonist administration to humans. The study shows the involvement of IL-10 in the pathogenesis of SLE, and indicates that the use of IL-10 antagonists may be beneficial in the management of refractory SLE.

Quantification of regulatory T cells in patients with systemic lupus erythematosus

CD4+ T cells that constitutively express CD25 exhibit powerful suppressive properties. Such cells have been denominated regulatory T cells (T(R)). Alterations in T(R)cells are known to cause organ-specific autoimmune disease in animal models. The aim of this work was to quantify CD4+CD25+ T cells in patients with systemic lupus erythematosus (SLE). Thirty untreated patients (ten with active disease) and ten healthy volunteers were studied. Flow cytometry was used to quantify cell populations. CD4+CD69+, CD4+CD25+ and CD4+CD25(bright) cells were considered. Peripheral blood mononuclear cell cultures were performed and supernatants collected for IL-10 and 12 measurement. CD4+CD25+ cells were significantly decreased in patients with active disease when compared to control subjects and patients without disease activity (P<0.001). CD4+CD69+ cells were increased in patients with active disease when compared to controls (P=0.041). Accordingly, CD4+CD25(bright) cells were decreased in patients with active disease compared to healthy subjects (P<0.001). IL-12 production was hampered in cells from patients during periods of active disease when compared to healthy controls and patients during remission (P<0.001). We observed a correlation between decreased T(R)number and reduced IL-12 mononuclear cell production (r=0.362, P=0.05). This work demonstrates that CD4+CD25+ T cells are decreased in patients with clinically active SLE.

Decreased production of and response to interleukin-2 by cultured lymphocytes from patients with systemic lupus erythematosus.

We studied the production of and response to interleukin-2 (IL-2) by peripheral blood T lymphocytes from 19 systemic lupus erythematosus (SLE) patients who received no treatment at the time they were studied. Eight had active disease and the rest were in remission. Results were compared with those obtained in 12 healthy subjects of similar age. T cells from SLE patients, whether activated with phytohemagglutinin or in autologous mixed lymphocyte reactions, were found to yield little IL-2, to have a low response to IL-2 from its own, or other sources, and to absorb IL-2 poorly, IL-2 produced by SLE cells, albeit scant, was absorbed normally by activated T cells from normal subjects. Our findings may contribute to the understanding of the immunoregulatory defect in SLE.

Quantitative and qualitative normal regulatory T cells are not capable of inducing suppression in SLE patients due to T-cell resistance

Previous reports have suggested that regulatory T cells (Treg) are abnormal in patients with systemic lupus erythematosus (SLE). In the present work, we quantified CD4+FOXP3+ Treg cells in patients with SLE and found no quantitative alterations. However, we found a clear defect in suppression assays. Surprisingly, SLE-derived Treg cells exhibited a normal phenotype and functional capacity. Conversely, SLE-derived CD4+CD25− effector T cells resisted suppression by autologous and allogeneic regulatory cells. Our findings strongly suggest that the defect in T-cell suppression observed in SLE is because of effector cell resistance and not because of an abnormal regulatory function.

Interleukin-1 and Interleukin-6 Activities are Increased in the Cerebrospinal Fluid of Patients with CNS Lupus Erythematosus and Correlate with Local Late T-Cell Activation Markers

We examined cerebrospinal fluid (CSF) samples from 12 patients with SLE and active central nervous system (CNS) involvement for their levels of the following cytokines: interleukin- 1 (IL-1) by means of two different assays-the IL-1 responsive murine cell line LBRM 33-1a5 and an ELISA for IL-1 alpha; IL-2 by means of the CTLL cell line responsive to it; and interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) both determined by a specific ELISA. We found that SLE CSF had significantly higher levels of IL-1 and IL-6 than did those obtained at surgery from eight controls without inflammatory neurologic disease. IL-2 and TNF were not detectable in any of the CSF samples. We also studied the status of activation in CSF T cells using monoclonal antibodies against early (anti-IL-2R (CD25) and antitransferrin (CD71)), late (anti-T10) and very late (anti-VLA-1) activation antigens, and found increased percentages of T10-bearing (18 ± 2 vs 3 ± 0.7%) and VLA-1-bearing T cells (12 ± 2 vs 0.7 ± 0.2%) in SLE patients as compared to controls (both P < 0.01). Levels of IL-1 and IL-6 correlated with T10 and those of IL-1 correlated also with VLA-1. Markers of early T-cell activation did not differ in SLE and control CSF. Because of these findings we analysed the effect of recombinant IL,-I, IL-6 or normal CSF on normal T cells and found that they did not induce the expression of activation markers. However, incubation in SLE CSF caused CD25, T10 and VLA-1 activation markers to become significantly expressed on cultured normal T cells. This expression of T10 and VLA-1 was partially inhibited by pre-incubation in anti-human IL-1 alpha polyvalent antibody. Our findings suggest increased in situ production of IL- and IL-6 and perhaps other factors in CNS lupus that might condition T-cell activation in the CNS compartment. These findings could have pathogenetic significance.

Cytokine gene expression in cirrhotic and non-cirrhotic human liver

BACKGROUND/AIMS In order to explore the role of cytokines in the pathogenesis of liver cirrhosis, we analyzed their gene expression in hepatic biopsies from patients with alcoholic liver cirrhosis, post-hepatitis C liver cirrhosis, and with idiopathic portal hypertension without cirrhosis. METHODS We assessed the gene expression of interleukins 1 beta, 2, 6, 8, and 10, as well as of tumor necrosis factor-alpha, transforming growth factor-beta and interferon-gamma by a quantitative polymerase chain reaction. RESULTS We found high levels of transforming growth factor-beta in post-hepatitis C liver cirrhosis, high to moderate in alcoholic liver cirrhosis and low in non-cirrhotic specimens. Expression of interleukin-10, tumor necrosis factor-alpha, and interferon-gamma genes was detected in most post-hepatitis C liver cirrhosis, but not in idiopathic portal hypertension or alcoholic liver cirrhosis biopsies. The interleukin1-beta, 6 and 8 gene expression was significantly lower in alcoholic liver cirrhosis compared to post-hepatitis C liver cirrhosis, but higher compared to idiopathic portal hypertension specimens. Thus, post-hepatitis C liver cirrhosis samples showed a high degree of cytokine gene expression, whereas in alcoholic liver cirrhosis it tended to be moderate, and restricted to some cytokines (transforming growth factor-beta, interleukin-1, 6 and 8, but not interleukin-10, tumor necrosis factor-alpha or interferon-gamma). In contrast, most non-cirrhotic specimens showed a restricted and low cytokine gene expression. CONCLUSIONS These data suggest that transforming growth factor-beta may have an important role in liver fibrosis and inflammation. Interleukin-1 beta, 6, 8, tumor necrosis factor-alpha and interferon-gamma, appear to participate in the pathogenesis of the mild to severe inflammatory phenomena seen in alcoholic and post-hepatitis C liver cirrhosis, respectively. Our data suggest that tumor necrosis factor-alpha does not participate in the hepatocellular damage of alcoholic liver cirrhosis, and indicate that neither interferon-gamma nor interleukin-10, at least at the levels observed in post-hepatitis C liver cirrhosis, are able to counteract the fibrotic/inflammatory process seen in this condition.

Adult-onset Still disease as the cause of fever of unknown origin.

Abstract: We conducted the current study to evaluate the cases of fever of unknown origin (FUO) admitted in our institution during the 10 years between 1991 and 2001 and to compare the patients diagnosed as having adult-onset Still disease (AOSD) with the patients with FUO due to other diagnoses. We performed a case-control study and analyzed 26 patients with AOSD and 135 patients with FUO due to other diseases. Controls were classified into 1 of 4 groups: 1. Infectious diseases; 2. Malignant conditions; 3. Autoimmune diseases; 4. No diagnosis. Differences between groups were evaluated by analysis of variance (ANOVA). Odds ratios (OR) were calculated by multiple logistic regression analyses. Patients with AOSD were younger than controls. Arthritis (OR, 8.6; 95% confidence interval [CI], 1.5-49.1; p = 0.014), pharyngitis (OR, 6.9; 95% CI, 1.5-30.2; p = 0.010), splenomegaly (OR, 5.4; 95% CI, 1.1-26.7; p = 0.039), and neutrophilic leukocytosis (OR, 18.1; 95% CI, 3.5-93.6; p = 0.001) were significantly more common in patients with AOSD than in the control groups. A clinical scale that identifies patients with AOSD was designed. It proved to be highly specific (≈98%), with predictive values greater than 90%. AOSD is a defined clinical entity. In most cases, it is clinically distinguishable from other causes of FUO. We propose a clinical scale as a tool to identify patients whose disease can be diagnosed based on clinical grounds without the need of long, costly diagnostic procedures. Abbreviations: AOSD = adult-onset Still disease, FUO = fever of unknown origin.

T-cell dysregulation in patients with hyperprolactinemia: Effect of bromocriptine treatment

We studied four patients with tumoral hyperprolactinemia and normal ovarian function before and after prolactine levels had become normal with treatment with bromocriptine (BrC), a dopamine agonist that inhibits prolactin release. Their proliferative responses to concanavalin A, pokeweed mitogen, and, to a lesser extent, phytohemagglutinin, their spontaneous and concanavalin A-induced suppression, and their production of interleukin 2 were found to be decreased and to correct partially or completely after bromocriptine treatment. The T-cell response to interleukin 2 was low in two patients in whom it increased after BrC treatment. These findings give insight on the immunomodulatory role of prolactin in vivo.

CTLA-4 and autoimmunity: New insights into the dual regulator of tolerance

Cytotoxic T-Lymphocye Antigen 4 (CTLA-4) or CD152 is an inhibitory molecule that plays a critical role in maintenance of tolerance to self-antigens. CTLA-4 is structurally as well as functionally related to CD28, since it shares 31% of homology and binds the B7 family molecules CD80 and CD86 with higher affinity. Nevertheless, CTLA-4 has opposing effects on T cell activation and current evidence shows that its inhibitory role goes beyond the ligand-binding interaction. CTLA-4 competes with CD28 in binding to B7, interacts within the immunological synapsis elements and with clathrin adaptor proteins and tyrosine phosphatases through its cytoplasmic domain to regulate cell trafficking and to set the activation threshold within T cells. Moreover, we have learned from the knock out model that CTLA-4 plays a key role in regulatory T cells and in central tolerance. Because of its importance in maintenance of peripheral tolerance, CTLA-4 has been implicated in several autoimmune diseases, such as systemic lupus erythematosus. Multiple single-nucleotide polymorphisms have been located to human Ctla-4 gene, and their association with autoimmune disease is still a matter of controversy. Despite the promising results of abatacept or CTLA-4-Ig in rheumatoid arthritis and murine lupus nephritis, more clinical randomized trials and standardization of outcomes are needed to prove its efficacy and safety in human lupus nephritis.