Laurie S. Davis

Combined Deficiency of Proapoptotic Regulators Bim and Fas Results in the Early Onset of Systemic Autoimmunity

Alterations in the stoichiometric balance between members of Bcl-2 and Fas apoptotic pathway could lead to the pathogenesis of systemic lupus erythematosus (SLE). We showed that patients with SLE displayed increased expression in antiapoptotic members of the Bcl-2 and Fas apoptotic pathways in isolated mononuclear cells. Further, mice (Bcl2l11(-/-)Fas(lpr/lpr)) lacking the Bcl-2 pro-apoptotic member, Bim (Bcl2l11(-/-)) and and with an lpr mutation in the gene encoding Fas (Fas(lpr/lpr)) developed severe SLE-like disease by 16 weeks of age unlike Bcl2l11(-/-) or Fas(lpr/lpr) mice. Bcl2l11(-/-)Fas(lpr/lpr) antigen-presenting cells (APCs) were markedly activated, and their numbers were increased in lymphoid tissues and in kidneys, yet numerous TUNEL-positive cells were observed in glomeruli of Bcl2l11(-/-)Fas(lpr/lpr) mice. These data demonstrate that dysregulation of the Bcl-2 or Fas pathways can alter the function of APCs, thereby leading to SLE pathogenesis.

Accessory Cell Signals Involved in T‐Cell Activation

The physiologic recognition of antigen by T lymphocytes involves an interaction of the T-cell receptor (TCR) with fragments of antigen bound to either class I or class II major histocompatibility complex (MHC) molecules on the surface of an antigen-presenting cell (APC) (Buus et al. 1986, 1987, Babbitt et al. 1985, Ohashi et al. 1985). It is felt that activation signals generated as a result of recognition of the antigen-MHC complex by the TCR are transmitted via CD3, a multimolecular complex expressed in association with the TCR on the surface of all mature T cells. The CD3 moleeular complex has a number of features characteristic of signaling molecules, including polypeptide members with substantial intracytoplasmic domains containing amino acid residues that are phosphorylated after stimulation (Samelson et al. 1985a, 1985b). That CD3 serves to convey signals to the T-ee!l upon antigen recognition is derived from the observation that the TCR is structurally, albeit not covalently, associated with the CD3 molecular complex (Brenner et al. 1987), whereas the structure of the TCR itself has little in common with other known signaling molecules (Hedrick et al. 1984, Sim & Augustin 1985). Moreover, CD3 and the TCR are coordinately expressed on T cells (Weiss & Stobo 1984). In addition, monoclonal antibodies (mAb) to CD3 trigger T-cell activation {Chang et al. 1981, Van-Wauwe et al. 1980). Finally, expression of CD3 is required for the activation of T cells stimulated via a number of different surface molecules (Bockenstedt et al. 1988, Bamezai et al. 1988, Gunter et al. 1987, Fleischer et al. 1988, Geppert et al. 1989). These results all support the conclusion that the CD3 molecular complex plays a eentral role in the transduction of signals from a variety of receptors including the TCR that culminate in T-cell activation after antigen recognition.

The role of cytokines in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by persistent synovitis, local destruction of bone and cartilage and many systemic manifestations. Chronic inflammation within the synovial tissue appears to be the basis of all these manifestations of RA [24, 101]. Although the etiologic stimulus has not been identified, established rheumatoid synovitis is characterized by persistent immunologic activity. The predominant infiltrating cell in the rheumatoid synovium is the T lymphocyte [181]. T4 (helper-inducer) cells predominate over T8 (suppressor-cytotoxic) cells and are frequently found in close proximity to HLA-DR-positive macrophages and dendritic cells. Analysis of T cells in synovial fluid has documented an enrichment in CD29 expressing memory T4 cells and a marked reduction in the number of CD45R expressing naive T4 cells [120]. The infiltrating T cells appear to be activated, since they express activation antigens, such as HLA-DR [23]. In addition, they express an increased density of molecules, such as leukocyte functionassociated antigen 1 (LFA-1, CD1 la/CD18) that have been implicated in a variety of cell to cell interactions, including binding of circulating cells to postcapillary venules just prior to entry into sites of tissue inflammation. Finally, the T cells appear to have proliferated locally in the synovial tissue, perhaps in response to sequestered antigen, since they express determinants such as very late antigen (VLA-1) that appears on T cells only after prolonged proliferation [23]. Evidence of B cell activation can also be found in the inflamed synovium. Activated B lymphoblasts and plasma cells producing immunoglobulin and rheumatoid factor are characteristic features of rheumatoid synovitis [186]. Large numbers of macrophages with an activated phenotype are also found in rheumatoid synovium [43]. As a site of persistent inlmunologically mediated inflammation, the rheumatoid synovium is characterized by the presence of a number of secreted products of

Oxidative Stress Promotes Polarization of Human T Cell Differentiation Toward a T Helper 2 Phenotype

These studies were conducted to determine the effects of oxidative stress on human T cell differentiation and polarization into Th1 or Th2 phenotypes. Highly purified naive CD4+ T cells were isolated from PBMC of healthy, nonatopic donors. CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence or absence of oxidative stress as supplied by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates a low level of superoxide anion. Increases in cellular superoxide were observed by exposure to DMNQ. Exposure of unpolarized CD4+ T cells to IL-12 or IL-4 resulted in a Th1 or Th2 phenotype, respectively. T cells stimulated in the absence of polarizing cytokines secreted modest amounts of IFN-γ and TNF-α. Cells stimulated in the continuous presence of 5 μM DMNQ, displayed a marked up-regulation in Th2 cytokines, including IL-4, IL-5, and IL-13, but not the Th1 cytokine IFN-γ. Th2 responses were blunted by concomitant exposure to thiol antioxidants. Long-term exposure of T cells to DMNQ resulted in growth of cells expressing CCR4, and a decrease in cells expressing CXCR3, indicating phenotypic conversion to Th2 cells. These results suggest that oxidative stress favors a Th2-polarizing condition.

SLE Peripheral Blood B Cell, T Cell and Myeloid Cell Transcriptomes Display Unique Profiles and Each Subset Contributes to the Interferon Signature

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19+ B lymphocytes, CD3+CD4+ T lymphocytes and CD33+ myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33+ myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3+CD4+ T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.

Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein.

Recent evidence suggests that B‐ and T‐cell interactions may be paramount in relapsing‐remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B‐cell pools from RRMS patients may specifically harbor a subset of potent neuro‐APC that support neuro‐Ag reactive T‐cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA‐DR expression, IL‐10 and lymphotoxin‐α secretion, neuro‐Ag binding capacity, and neuro‐Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4+ T‐cell proliferation and IFN‐γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4+ T‐cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B‐cell pool in RRMS harbors neuro‐Ag specific B cells that can activate T cells.

Shared signaling networks active in B cells isolated from genetically distinct mouse models of lupus

Though B cells play key roles in lupus pathogenesis, the molecular circuitry and its dysregulation in these cells as disease evolves remain poorly understood. To address this, a comprehensive scan of multiple signaling axes using multiplexed Western blotting was undertaken in several different murine lupus strains. PI3K/AKT/mTOR (mTOR, mammalian target of rapamycin), MEK1/Erk1/2, p38, NF-kappaB, multiple Bcl-2 family members, and cell-cycle molecules were observed to be hyperexpressed in lupus B cells in an age-dependent and lupus susceptibility gene-dose-dependent manner. Therapeutic targeting of the AKT/mTOR axis using a rapamycin (sirolimus) derivative ameliorated the serological, cellular, and pathological phenotypes associated with lupus. Surprisingly, the targeting of this axis was associated with the crippling of several other signaling axes. These studies reveal that lupus pathogenesis is contingent upon the activation of an elaborate network of signaling cascades that is shared among genetically distinct mouse models and raise hope that targeting pivotal nodes in these networks may offer therapeutic benefit.

Expansion of activated human naïve T‐cells precedes effector function

Naïve T‐cells divide and mature, both functionally and phenotypically, upon stimulation through the T‐cell receptor. Although much is known about the overall changes that occur in naïve cells upon TCR stimulation, and the different memory/effector populations that arise following stimulation, the relationship between cell division and functional and phenotypical changes that occur after activation is poorly understood. Here, we examine the early stages of human naïve and antigen‐experienced T‐cell activation, and the relationship between cell division and acquisition of effector function during the transition from resting antigen‐experienced or naïve T‐cells into effector cells. Stimulated naïve T‐cells proliferate prior to acquisition of effector function, as measured by cytokine production and expression of effector‐associated cell surface molecules. Additionally, we show that interlukin‐7 (IL‐7) can drive proliferation of naïve T‐cells without TCR:MHC peptide interactions. IL‐7 alone does not, however, drive the proliferation of antigen‐experienced T‐cells. Memory T‐cells will divide in response to exogenous IL‐7 but only in the presence of naïve T‐cells and IL‐2. This study contributes to the current understanding of the mechanistic differences between naïve and memory T‐cell responses by defining the functional and phenotypic changes that occur to T‐cells after stimulation.

The Role of Cytokines in the Pathogenesis and Treatment of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by a defect in immune tolerance and exacerbated by both the innate and adaptive arms of the immune response. SLE-associated immune hyperactivity can be detected systemically as elevations in levels of cytokines along with their upregulated receptors expressed by hematopoietic cells. Importantly, increased levels of cytokines and their receptors can be observed in target organs, and it is clear that they have important roles in disease pathogenesis. Recent therapeutic strategies have focused on proximal cytokines, such as interferon-α, interleukin (IL)-1, IL-6, and tumor necrosis factor as a result of the efficacious use of biologic agents for intervention in rheumatoid arthritis and other autoimmune diseases. Despite the recent advances in understanding the cytokine networks involved in autoimmune diseases and more specifically in SLE, the diagnosis and prognosis of lupus remain a challenge. Lupus is heterogeneous and unpredictable; moreover, the frequency and severity of flares can be difficult to determine and treat. A better understanding of the regulation of expression of key cytokines and their receptors can likely provide important clues to the pathogenic mechanisms underlying specific forms of SLE, and pave the way toward more effective therapeutics.

Dysregulated expression of CXCR4/CXCL12 in subsets of patients with systemic lupus erythematosus.

OBJECTIVE CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up-regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE. METHODS Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti-CXCL12 antibody. RESULTS CD19+ B cells and CD4+ T cells from SLE patients displayed a >2-fold increase (P = 0.0001) and >3-fold increase (P < 0.0001), respectively, in median CXCR4 expression compared with that in controls (n = 7-8). Moreover, CXCR4 expression on B cells was 1.61-fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n = 8) than in patients with SLEDAI scores ≤10 (n = 16) (P = 0.0008), 1.71-fold higher in patients with class IV LN (n = 5) than in patients with other classes of LN (n = 7) (P = 0.02), and 1.40-fold higher in patients with active neuropsychiatric SLE (NPSLE) (n = 6) than in patients with inactive NPSLE (n = 18) (P = 0.01). CXCL12 was significantly up-regulated in the tubules and glomeruli of kidneys in patients with LN (n = 14), with the percentage of positive cells correlating positively with the severity of LN. CONCLUSION CXCR4 appears to be up-regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.