The sphingosine-1-phosphate (S1P) lysophospholipid receptor S1P3 regulates MAdCAM-1+ endothelial cells in splenic marginal sinus organization

Marginal zones (MZs) are microdomains in the spleen that contain various types of immune cells, including MZ B cells, MOMA1(+) metallophilic macrophages, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)(+) endothelial cells. MAdCAM-1(+) and MOMA1(+) cells line the sinus, that separates MZs from splenic follicles. Here we show that a receptor for the lysophospholipid sphingosine-1-phosphate (S1P), S1P(3), is required for normal numbers of splenic immature and MZ B cells, and for S1P-induced chemotaxis of MZ B cells. S1P(3) is also essential for proper alignment of MOMA1(+) macrophages and MAdCAM-1(+) endothelial cells along the marginal sinus. The lack of cohesion of the marginal sinus in S1P(3)(-/-) mice affects MZ B cell functions, as wild-type (WT) MZ B cells migrate more into S1P(3)(-/-) follicles than into WT follicles after treatment with lipopolysaccharide. Additionally, short-term homing experiments demonstrate that WT MZ B cells home to the S1P(3)(-/-) spleen in increased numbers, suggesting a role for the marginal sinus in regulating MZ B cells numbers. Moreover, S1P(3)(-/-) mice are defective in mounting immune responses to thymus-independent antigen type 2 due to defects in radiation-resistant cells in the spleen. These data identify lysophospholipids and the S1P(3) receptor as essential regulators of the MZ sinus and its role as a barrier to the follicle.     

The Lsc RhoGEF mediates signaling from thromboxane A2 to actin polymerization and apoptosis in thymocytes

The Lsc RhoGEF (also known as p115-RhoGEF) is a GTP exchange factor (GEF), an activator of GTPases of the Rho family. Lsc has a RhoGEF domain specific for Rho GTPase and a regulator of G protein signaling (RGS) domain specific for Galpha(12/13) subunits. One G protein receptor that can couple to Galpha(12/13) subunits is the receptor for thromboxane A(2 )(TXA(2)), thromboxane-prostanoid (called TP), which is highly expressed in immature thymocytes. TXA(2) has been implicated in thymocyte apoptosis. We found that Lsc(-/-) mice on a BALB/c background show thymic hyperplasia due to increased numbers of thymocytes and that these numbers further increase with the age of the mice. To investigate a role for Lsc in TXA(2) signaling, we analyzed activation of primary thymocytes by TXA(2) in vitro. TXA(2)-induced apoptosis of double-positive thymocytes and Rho activation required Lsc, and TXA(2) stimulation of actin polymerization and cofilin phosphorylation required both Lsc and Rho kinase (ROCK). Additionally, in the absence of Lsc, phosphorylation of the survival kinase Akt in response to TXA(2) was greatly enhanced. Together, these data demonstrate that Lsc is essential for mediating TXA(2 )signaling involved in apoptosis and actin organization and suggest that TXA(2) regulates thymic cellularity via Lsc.     

Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling

Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor-induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1(-/-)Vav-2(-/-) B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-independent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1(-/-)Vav-2(-/-) B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.     

Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production

Objective. To investigate whether histone-specific T helper (Th) cells that are able to induce anti-doublestranded DNA (anti-dsDNA) antibodies can be isolated from patients with systemic lupus erythematosus (SLE) and to characterize the cytokine secretion pattern of such Th clones. Methods. Peripheral blood mononuclear cells from SLE patients and healthy donors were stimulated with autologous apoptotic cell material or purified histones, expanded with interleukin-2 (IL-2), and cloned by limiting dilution. Histone reactivity of clones was examined by histone-specific proliferation and cytokine release. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Induction of anti-dsDNA antibodies was measured in cocultures of autologous B cells and Th clones by ELISA. Results. Numerous histone-specific T cell receptor (TCR) α/β+ Th clones were established from 2 of 3 patients with active SLE and from 1 of 2 healthy individuals. Most Th clones secreted IL-2, interferon-γ (IFNγ), and IL-4, whereas some produced predominantly IL-2 and lFNγ. Th clones that could stimulate the production of anti-dsDNA antibodies were derived from SLE patients and from a healthy individual. Conclusion. Th cells specific for histones may play an important role in the pathogenesis of SLE by inducing autoantibodies to dsDNA. Both Th1 and Th2 cytokines may be involved in the pathogenesis of SLE. The presence of histone-specific Th cells in a healthy individual indicates the importance of peripheral tolerance for preventing autoimmunity to nuclear antigens.     

Apoptotic cells selectively suppress the Th1 cytokine interferon γ in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-β, but less of the pro-inflammatory cytokines TNFα, IL-1β and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferonγ (IFNγ) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

Apoptotic cells selectively suppress the Th1 cytokine interferon gamma in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-beta, but less of the pro-inflammatory cytokines TNFalpha, IL-1beta and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferon gamma (IFN gamma) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

Involvement of phosphatidylserine, alphavbeta3, CD14, CD36, and complement C1q in the phagocytosis of primary necrotic lymphocytes by macrophages

OBJECTIVE: Uningested dead cells may be an important source of autoantigens and may trigger autoimmune diseases such as systemic lupus erythematosus (SLE). Multiple receptors involved in the clearance of apoptotic cells have been described; however, little is known about the receptors and ligands involved in uptake of necrotic cells that release autoantigens as well. METHODS: The uptake of autologous necrotic peripheral blood lymphocytes into human monocyte-derived macrophages was qualitatively and quantitatively monitored by confocal microscopy and 2-color flow cytometry, respectively. Blocking experiments were performed to examine the receptors and molecules involved in the phagocytosis of necrotic cells. Cytokine secretion by lipopolysaccharide-activated monocytes and macrophages was determined by enzyme-linked immunosorbent assay. RESULTS: Phosphatidylserine, which was exposed on necrotic as well as apoptotic cells, promoted the recognition and removal of primary necrotic lymphocytes. Several macrophage receptor systems, including the thrombospondin-CD36-alphavbeta3 complex, CD14, and the complement component C1q, contributed to the engulfment of necrotic cells. Necrotic peripheral blood lymphocytes slightly increased the lipopolysaccharide-induced secretion of interleukin-10 and reduced the secretion of tumor necrosis factor alpha in monocytes and macrophages. CONCLUSION: Our results indicate that at least some of the receptors and adaptors mediating the uptake of apoptotic cells are also involved in the clearance of necrotic cells. Hence, necrotic cells engage phagocyte receptors such as CD36, which mediate antiinflammatory signals from apoptotic cells. Necrotic cells consequently also have the potency to provide antiinflammatory signals to phagocytes; however, these signals may be overridden by proinflammatory factors released during necrosis. These findings have implications regarding the etiopathogenesis of autoimmune diseases such as SLE, in which impaired clearance of dead cells may foster autoimmunity by the release of potential autoantigens.     

Vav1-deficient mice are resistant to MOG-induced experimental autoimmune encephalomyelitis due to impaired antigen priming

Mice that lack the guanine nucleotide exchange factor (GEF) Vav1 exhibit particular defects in antigen-triggered T cell activation but may have an autoreactive T cell repertoire due to impaired intra-thymic negative selection. MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE) was used to test the susceptibility of Vav1(-/-) mice to organ-specific autoimmunity. Vav1(-/-) animals were found to be resistant to MOG(35-55)-EAE since the priming and in vivo expansion of myelin oligodendrocyte glycoprotein (MOG)-specific T cells was inefficient despite fully functional antigen presentation. Protection from cell-mediated autoimmunity was not due to a Th2 bias, to the lack of IL-2 or a failure of Vav1(-/-) T cells in terms of chemotactic mobility.