Inhibition of terminal complement: a novel therapeutic approach for the treatment of systemic lupus erythematosus.

The importance of the complement system in the pathophysiology of systemic lupus erythematosus (SLE) is clear although individual complement components play very different roles in the disease process. Early complement proteins are critical in the clearance of immune complexes and apoptotic bodies, and their absence predisposes individuals to SLE. Conversely, activation of terminal complement is associated with exacerbations of disease and damage to tissues and organs, particularly in lupus nephritis. Monoclonal antibodies that specifically inhibit terminal complement activation while preserving the critical functions of the early complement cascade have now been developed. These antibodies target the C5 complement protein, blocking its cleavage and the subsequent generation of potent proinflammatory molecules. Anti-C5 therapeutics have recently been investigated in an animal model of SLE and in a Phase I single dose study in humans. The results of these studies and the multiple roles of complement in SLE are discussed.

Aprotinin and the systemic inflammatory response after cardiopulmonary bypass.

Cardiopulmonary bypass is associated with a systemic inflammatory response, a spectrum of pathophysiologic changes ranging from mild organ dysfunction to multisystem organ failure. Complications include coagulation disorders (bleeding diathesis, hyperfibrinolysis) from platelet defects and plasmin activation, as well as pulmonary dysfunction from neutrophil sequestration and degranulation. Diverse injuries are a consequence of multiple inflammatory mediators (complement, kinins, kallikrein, cytokines). Both plasmin and kallikrein amplify the inflammatory response by activating components of the contact activation system. The full-Hammersmith (high dose) of aprotinin, a serine protease inhibitor approved for reducing blood loss and transfusion requirements in cardiopulmonary bypass, inhibits kallikrein and plasmin, resulting in suppression of multiple systems involved in the inflammatory response. Specifically, inhibition of factor XII, bradykinin, C5a, neutrophil integrin expression, elastase activity, and airway nitric oxide production are observed. Clinical correlates include reduced capillary leak, preserved systemic vascular resistance and blood pressure, and improved myocardial recovery following ischemia. Overall, evidence indicates that aprotinin attenuates the systemic inflammatory response associated with cardiopulmonary bypass.

Co-ligation of alpha4beta1 integrin and TCR rescues human thymocytes from steroid-induced apoptosis.

Maturation of thymocytes represents a sequence of events during which thymocytes expressing TCR with moderate avidity for self antigen/MHC are positively selected, whereas those with high or insufficient TCR avidity die. Glucocorticoids are produced intrathymically and can contribute to apoptosis of unselected thymocytes. Thymocytes differentiate in a close contact with epithelial cells, expressing vascular adhesion molecule-1 (VCAM-1) and secreting glucocorticoids, with bone marrow-derived macrophages, and with extracellular matrix containing fibronectin (FN) and collagen. Their contact with FN is mediated by alpha4beta1 and alpha5beta1 integrins. We examined the contribution of TCR and integrin signaling to the survival of thymocytes from dexamethasone (Dex)-induced apoptosis. We demonstrate that FN and VCAM-1 (both of which bind alpha4beta1 integrin), but not collagen, considerably augment TCR-mediated protection of thymocytes from Dex-induced apoptosis. This 'survival' signal is transduced through the alphabeta1, but not through the alpha5beta1 integrin. The observed protection from Dex-induced apoptosis correlated with an increase in bcl-2 protein levels. FN-alpha4beta1 and VCAM-1-alpha4beta1 engagement induced up-regulation bcl-2 protein, while alpha5beta1 binding to FN induced a negative signal that was blocked by anti-alpha5beta1 antibody. These data suggest that alpha4beta1 integrin may contribute to protection of thymocytes with moderate avidity TCR from glucocorticoid-induced death during intrathymic maturation.

Vascular cell adhesion molecule-1 is expressed by cortical thymic epithelial cells and mediates thymocyte adhesion. Implications for the function of alpha4beta1 (VLA4) integrin in T-cell development.

T-cell development requires a series of discrete selection and activation signals delivered to maturing progenitors in the thymic cortex and medulla. We have previously shown the constitutive activity of the integrin, alpha4beta1 (VLA4), on a unique subpopulation of immature cortical thymocytes and proposed a role for integrin-mediated adhesion in positive selection by cortical epithelium. In the present report we show that thymic epithelial cell lines express vascular cell adhesion molecule-1 (VCAM-1) a high-affinity ligand for apha4beta1, and that VCAM-1 mediates thymocyte binding to these lines. Immunohistochemistry and confocal microscopy show that VCAM-1 is selectively expressed in situ by thymic epithelium in the cortex and corticomedullary junction, two locations at which VCAM-1 could determine the interaction between immature thymocytes and selecting elements on epithelial cells. In parallel, we confirmed that fibronectin (FN), the alternative ligand for alpha4beta1, is expressed predominantly in the medulla. These results suggest that VCAM-1 is an adhesive ligand in the thymic cortex for the activated form of alpha4beta1 constitutively expressed during development by immature double positive thymocytes. The structural segregation of the alternative ligand, FN, to the medulla suggests that medullary FN may regulate the migration, development, and export of more mature thymocytes.

End-stage renal disease and systemic lupus erythematosus.

OBJECTIVE: To provide an overview of the course of systemic lupus erythematosus (SLE) following the onset of end-stage lupus nephropathy, regarding clinical and serological manifestations, survival on dialysis, and renal transplant outcomes. METHODS: A review of the pertinent literature, identified by a comprehensive Grateful Med search, was performed. RESULTS: There is a tendency for decreased clinical and serological lupus activity following the onset of end-stage renal disease. The pathophysiology of this quiescence remains unclear. Survival of lupus patients on dialysis is no different from that of non-SLE dialysis patients, and is better than that of several other rheumatic diseases. Following renal transplantation, there is no difference in patient or graft survival in lupus versus nonlupus patients. Like their nonlupus counterparts, SLE transplant patients do better with living relative grafts and/or regimens containing cyclosporin A. Transplantation is not recommended within 3 months of the initiation of dialysis to allow possible recovery from the acute renal failure. Transplantation during an acute exacerbation of SLE is controversial, and may increase the risk of poor outcomes. Recurrence of lupus in transplanted allografts, often with the same histopathology as in the native kidney, occurs at a rate (2.7% to 3.8%) comparable to that for all allograft transplant failures (2% to 4%). CONCLUSIONS: End-stage lupus nephropathy patients require less medication owing to decreased disease activity. They are good candidates for dialysis and renal transplantation, with survival and recurrence rates no different from those of other patients with end-stage renal disease.

Differential expression of integrins on human thymocyte subpopulations.

Integrins represent a candidate group of cell surface receptors that may control the homing and population of the thymus by T-cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. We have used multiparameter flow cytometric methods to characterize the expression of several members of the integrin family (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha L beta 2) on thymocyte subpopulations and have correlated integrin expression with other well-defined thymocyte differentiation markers. alpha 4 beta 1 was expressed by all thymocytes, but expression was highest on CD4-CD8- double-negative (DN) cells, high on CD+CD8+ double-positive (DP) cells, and lowest on mature single-positive (SP) cells, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were present on 13%, 63%, and 26% of thymocytes, respectively, with maximal levels of expression on DN and SP cells, and low levels of expression on DP cells. Simultaneous analysis of alpha 4 beta 1, alpha 5 beta 1, and CD3 expression suggested a pathway of T-cell differentiation in the thymus in which the majority of the DN cells were alpha 4 beta 1hi alpha 5 beta 1hi, the DP cells alpha 4 beta 1hi alpha 5 beta 1lo/-, and the most mature SP cells were alpha 4 beta 1int. The stage-specific expression of integrins strongly implies their functional involvement during T-cell maturation in the thymus.

Alpha 3 beta 1 and alpha 6 beta 1 integrins mediate laminin/merosin binding and function as costimulatory molecules for human thymocyte proliferation.

Integrins comprise a superfamily of alpha beta heterodimers that serve as cell signaling as well as adhesion molecules. We demonstrate that the alpha 3 beta 1 and alpha 6 beta 1 integrins are laminin/merosin receptors expressed in human thymocytes. By reverse transcriptase-PCR analysis, we determined that the alpha 3A beta 1, but not the alpha 3B beta 1, cytoplasmic structural variant of alpha 3 beta 1 is expressed in thymocytes. In contrast, both alpha 6A beta 1 and alpha 6B beta 1 cytoplasmic structural variants of alpha 6 beta 1 are expressed. A small percentage (10 to 15%) of human thymocytes bind to immobilized laminin, and even fewer (3 to 5%) bind to merosin, the laminin isoform normally present in the thymus. This binding, however, can be increased to 39 to 41% after activation of thymocytes with Mn2+ (or PMA). Binding to either laminin or merosin is completely inhibited by anti-beta 1 mAb or by a mixture of anti-alpha 3 and anti-alpha 6 mAbs, indicating that both alpha 3 beta 1 and alpha 6 beta 1 participate in thymocyte adhesion to the laminin family of extracellular matrix proteins. The protein kinase C inhibitors, calphostin C and staurosporine, inhibit Mn(2+)-enhanced thymocyte binding, suggesting that protein kinase C activity is crucial for the binding. Furthermore, the data indicate that at least two divalent cation binding sites serve to regulate integrin binding activity. Finally, we show that both immobilized laminin and merosin have costimulatory function for anti-CD3-induced thymocyte proliferation, and both anti-alpha 3 and anti-alpha 6 mAbs can block this proliferative response. The cooperative function of alpha 3 beta 1 and alpha 6 beta 1 evidenced in the laminin/merosin binding and proliferation assays suggests that thymocyte-merosin interactions may play an important role in thymic T cell development.

Role of endogenous retroviruses in autoimmune diseases.

Retroviruses have been implicated in the pathogenesis of murine and human lupus; however, many positive findings have been followed by alternative explanations. Initial findings implicating xenotropic retroviruses were subsequently invalidated. The first solid demonstration that endogenous retroviruses mediate disease was the study of SL/Ni mice. Here budding ecotropic retroviral particles from arterial smooth muscle cells caused an antibody response to the particles with subsequent complement deposition. Our laboratory has focused on derangements in endogenous MCF retroviral expression. We found that lupus-prone NZB, BXSB and MRL strains have a marked increase in expression of Mpmv RNA in their thymuses while bone marrow expression did not differ from normal strains. Sequence analysis demonstrated mutations in the NZB endogenous retroviruses which could alter expression. A phosphorothioate antisense oligonucleotide to the initiation sequence of Mpmv caused lymphocyte activation in vivo in normal mice, providing further evidence for in vivo effects of Mpmv and potential for pathological abnormalities in lupus-prone strains.

Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development.

Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1-dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes.

Administration of a phosphorothioate oligonucleotide antisense to murine endogenous retroviral MCF env causes immune effects in vivo in a sequence-specific manner.

Previous in vitro studies had suggested that a product of the env gene of murine MCF (polytropic)-related sequences plays a role in regulating lymphocyte activation. To determine whether such an effect occurs in vivo, we have studied mice injected with phosphorothioate oligonucleotides antisense to such sequences. Injection of mice with antisense to the initiation region of the env gene resulted in (i) increased spleen cell numbers, primarily due to an increase in splenic B cells, (ii) increased class II MHC expression on B cells, (iii) increased RNA and DNA synthesis, and (iv) increased numbers of Ig producing cells. These results obtained with the antisense to MCF-related env did not occur with two scrambled phosphorothioate oligonucleotides or with antisense oligonucleotides to the initiation region of the env gene of xenotropic or ecotropic retroviral sequences. These data suggest that products of certain endogenous retroviral sequences regulate lymphocyte activation in vivo.

Molecular aspects of systemic lupus erythematosus: murine endogenous retroviral expression.

Systemic lupus erythematosus is an immune-mediated disease in which the etiology is unknown. Full-length (8.4 kb), type C, modified polytropic (Mpmv) retroviral transcripts from the thymus are characteristic of murine lupus. Reciprocal bone marrow transplantation studies determined that this thymic expression maps to the pre-T bone marrow stem cell. In vitro and in vivo oligonucleotide antisense work suggest that type C retroviruses play a role in immune activation. This paper summarizes our studies of endogenous retroviruses in murine lupus.

Characterization of RT6-bearing rat lymphocytes. II. Developmental relationships of RT6- and RT6+ T cells.

The derivation of RT6+ T cells from postthymic RT6- T cells in weanling rats was formally demonstrated by the intravenous transfer ("parking") of highly purified populations of RT6- lymph node T cells into thymectomized, irradiated, and bone-marrow-reconstituted (TXBM) RT6 and RT7 alloantigen-disparate recipients. Parallel experiments in irradiated and bone-marrow-reconstituted rats, and in rats whose RT6+ T cells had been depleted by injection of DS4.23 anti-RT6.1 mAb, suggested that the transit time between the pre-RT6+ and the RT6+ T-cell compartments approximated 4-5 days. A more precise estimate of the transit time was made by linear regression analysis of the generation of RT6+ T cells in rats that were treated with DS4.23 mAb at timed intervals after thymectomy. This study indicated that 50% of the pre-RT6+ T cells differentiated into RT6+ cells within 4 days, 75% within 8 days, and more than 90% within 16 days. Despite the apparent absence of pre-RT6- T cells 3 weeks after thymectomy, numerous RT6- T cells persisted for at least 10 weeks in thymectomized rats, even after treatment with DS4.23 mAb. Moreover, these RT6+ T cells failed to generate RT6+ T cells after transfer into adoptive hosts. Quantitative and phenotypic analyses indicated that this population of "true" RT6- T cells: (1) constitutes approximately 50% of the total RT6- T cells normally found in control rats; (2) contains CD4+ and CD8+ subsets; (3) expresses both the CD5 pan-T-cell antigen (which is absent from NK cells) and the R73 alpha/beta TCR constant-region determinant; and (4) lacks sIgM. Hence, the present results indicate that the "true" RT6- and the RT6+ T-cell subsets have stable antigenic phenotypes and represent developmentally discrete populations of postthymic cells in normal rats. This is supported by associated phenotypic and functional studies that suggest that the "true" RT6- T-cell subset contains antigenically naive and/or autoreactive clonotypes, whereas the RT6+ T-cell subset contains memory and/or regulatory cells. It remains to be determined whether the "true" RT6- and the RT6+ subsets represent separate lineages of T cells or a single lineage at different stages of activation or maturation.

Characterization of RT6 bearing rat lymphocytes. I. Ontogeny of the RT6+ subset.

The RT6 alloantigen is present on approximately 70% of peripheral T cells in the rat, but is absent from thymocytes and bone marrow lymphocytes. The results of further phenotypic analysis in the present study demonstrated that the RT6 alloantigen is expressed on approximately 45% of the helper/inducer (CD4; W3/25+) and 80% of the cytotoxic/suppressor (CD8; OX8+) peripheral T-cell subsets. Ontogenetic and thymus ablation studies indicated that the RT6+ T-cell subset is thymus-dependent and normally develops after the appearance of RT6-T cells in neonatal rats, and that the expression of RT6 is a post-thymic maturational event. Furthermore, intrathymic adoptive transfer of bone marrow cells demonstrated that RT6+ T cells are thymus-derived cells. These results show that most if not all RT6+ T cells are the progeny of RT6- T cells. However, they do not exclude the possibility that a separate lineage of RT6- T cells exists, which also has OX8+ and W3/25+ subsets. The possible developmental and functional relationships of RT6- and RT6+ T cells in the rat are discussed.

Monoclonal antibodies to RT7 and LCA antigens in the rat: cell distribution and segregation analysis.

Fluorescein-conjugated monoclonal antibodies (mAb) have been used to examine the cellular distribution and genetic relationship of the RT7 and the leukocyte common antigen (L-CA) in the rat. We demonstrate here that the RT7.1 alloantigen, recognized by the mAb BC84, is present on B lymphocytes as well as T lymphocytes, although at markedly different apparent concentrations; T cells binding approximately 4 times more BC84 than do B cells. In contrast, the polymorphic L-CA recognized by the NDS58 mAb and the non-polymorphic L-CA recognized by the OX1 mAb appear to be expressed in equal concentrations on T and B cells and differ in their tissue distribution from the RT7.1 alloantigen. We have also tested a new mAb termed 8G6.1, that is reactive with RT7b rat strains and has a cell, tissue and strain distribution profile resembling that of a polymorphic L-CA. Segregation analysis of the RT7 and polymorphic L-CA antigenic systems using a single backcross model demonstrate that the alloantigens recognized by BC84 and NDS58 cosegregate and that both are allelic with respect to 8G6.1. The L-CA antigenic determinant defined by OX1 was non-polymorphic and thus was not allelic with any of the antigenic systems tested. These results suggest that there is a close genetic relationship between the expression of RT7 and L-CA in the rat.

Target-cell recognition by cloned lines of influenza virus-specific cytotoxic T lymphocytes. Selective inhibition by a monoclonal H-2-specific antibody.

A monoclonal H-2d-specific antibody markedly inhibits target-cell lysis mediated by two influenza virus A/JAP/57-specific, H-2Kd-restricted cloned CTL lines. Three other A/JAP-57-specific, H-2d-restricted CTL clones (two of which are also restricted to H-2Kd in target-cell recognition) are only minimally inhibited by this monoclonal antibody. The inhibitory effect of the antibody is not due to selective binding to certain cloned CTL lines but rather is due to blocking of a determinant on the target cell. The monoclonal antibody produces partial inhibition of lysis mediated by a heterogeneous population of A/JAP/57-specific, H-2d-restricted CTL. Likewise the profound, selective inhibition of cytolysis produced by the H-2d-specific monoclonal antibody could not be reproduced with a conventional H-2d-specific alloantiserum. These observations suggest that more than one site on a particular H-2K or H-2D molecule can serve as a determinant for H-2-restricted CTL recognition. They furthermore imply that there is more than one recognition structure (receptor) for self MHC products clonally distributed among a population of H-2-restricted CTL directed to a particular antigen.