Serum Wisteria floribunda agglutinin-positive Mac-2-binding protein can reflect systemic lupus erythematosus activity.

Serum Mac-2-binding protein (M2BP) is elevated in various chronic inflammatory diseases, and evidence suggests that glycosylation of M2BP induces discrete biological effects. However, the role of serum M2BP in systemic lupus erythematosus (SLE) is still unclear. Recently, a Wisteria floribunda agglutinin-positive-M2BP (WFA+-M2BP) immunoassay has shown promise in detecting highly glycosylated M2BP. In this study, by using WFA+-M2BP immunoassay, we measured serum M2BP in 203 SLE patients and evaluated its clinical significance. Eighty patients were classified as having active SLE and 123 patients as having inactive SLE. The median serum M2BP was higher in patients with active SLE than in those with inactive SLE (2.1 vs. 0.9, p < 0.001). In multivariate linear regression analysis, serum M2BP, anti-dsDNA, C3 and erythrocyte sedimentation rate (ESR) were associated with SLEDAI-2K. Serum M2BP also strongly correlated with laboratory variables related to SLEDAI-2K, ESR and C-reactive protein. Furthermore, multivariate logistic regression analysis demonstrated that serum M2BP was useful in predicting active SLE. Finally, following immunosuppressive treatment, elevated serum M2BP significantly decreased along with improvement in disease activity. These findings suggest that serum M2BP might contribute to the inflammatory process in SLE, and measuring serum M2BP might be a useful marker to assess SLE disease activity.

Calcitonin induces dephosphorylation of Pyk2 and phosphorylation of focal adhesion kinase in osteoclasts.

Calcitonin induces the association and tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and HEF1 in HEK-293 cells that overexpress the calcitonin receptor (C1a-HEK), but the hormone's effect on these adhesion-related proteins in osteoclasts is not known. We therefore studied the effect of calcitonin on the tyrosine phosphorylation and subcellular distribution of paxillin, HEF1, FAK, and Pyk2, a FAK-related tyrosine kinase, in osteoclasts. Osteoclasts expressed both Pyk2 and FAK, with Pyk2 much more highly expressed. The two tyrosine kinases and paxillin were prominently associated with small punctate structures that were most densely clustered in the region of the peripheral F-actin-rich ring. Some of the punctate structures stained either for Pyk2 alone or FAK alone. Treatment with calcitonin disrupted the actin ring and induced the loss of the peripheral staining of paxillin, Pyk2, and FAK. In calcitonin-treated osteoclast-like cells, the tyrosine phosphorylation of paxillin and FAK increased, whereas the tyrosine phosphorylation of Pyk2 decreased. Calcitonin also induced increased phosphorylation of Erk1 and Erk2 in osteoclasts, as it did in the C1a-HEK cells. The unexpected dephosphorylation of Pyk2 correlated with decreased phosphorylation of Tyr(402), the autophosphorylation site of Pyk2. The calcitonin-induced dephosphorylation of Pyk2 was not observed in C1a-HEK cells transfected with Pyk2, suggesting that the reduced phosphorylation seen in osteoclasts may be specific to these cells. Treatment of osteoclast-like cells with 12-phorbol 13-myristate acetate increased the tyrosine phosphorylation of both Pyk2 and FAK, and calphostin C, an inhibitor of protein kinase C, blocked calcitonin-stimulated FAK phosphorylation. Increasing intracellular calcium with ionomycin caused a decrease in the tyrosine phosphorylation of Pyk2 and the loss of the actin ring in a manner similar to the effect of calcitonin. Ionomycin had no effect on FAK tyrosine phosphorylation. Calcitonin (CT)-induced changes in Pyk2, FAK, and Erk1/2 phosphorylation were independent of c-Src.

The Src-protein tyrosine kinase Lck is required for IL-1-mediated costimulatory signaling in Th2 cells.

Src-protein tyrosine kinases are intimately involved in TCR-initiated signaling in T lymphocytes. One member of this family, Lck, is also involved in CD28-mediated costimulation in Th1 cells. In Th2 lymphocytes, the costimulatory signal can also be provided by the interaction of IL-1 with type I IL-1R (IL-1RI), culminating in the activation of NF-kappaB transcription factors. Proximal steps in the IL-1R pathway, however, remain poorly understood, and there is conflicting evidence as to the importance of tyrosine phosphorylation in IL-1R signaling. We have addressed this issue by examining the ability of IL-1 to costimulate the activation of Lck-deficient Th2 cells. Our data demonstrate that, in the absence of Lck, the IL-1 costimulatory pathway is blocked despite the expression of normal levels of IL-1RI. Moreover, the block is associated with a defective degradation of IkappaB-alpha and an incomplete activation of NF-kappaB heterodimeric complexes. Protein expression of NF-kappaB monomers, including p50, p65, and c-Rel, is equivalent in both wild-type and Lck-deficient Th2 cell clones. Finally, we demonstrate that, in normal Th2 cells, stimulation with IL-1 leads to a rapid induction in tyrosine phosphorylation of several substrates including Lck itself. These findings strongly suggest that Lck is required for signaling in the IL-1 costimulatory pathway in Th2 lymphocytes.

Human T cells infiltrate and injure pig coronary artery grafts with activated but not quiescent endothelium in immunodeficient mouse hosts.

BACKGROUND: We have previously demonstrated that human artery grafts transplanted to immunodeficient mice are infiltrated and injured by unsensitized allogeneic human T cells. We extended our investigations to human anti-porcine xenoresponses in this model. METHODS: Pig coronary artery segments were interposed into the infrarenal aorta of severe combined immunodeficiency/beige mice. After 7 days, certain recipients were reconstituted with human leukocytes and/or treated with proinflammatory cytokines. The grafts were harvested after 1-70 days and examined by histology, immunohistochemistry, and morphometry. RESULTS: Pig artery grafts from untreated mice had no evidence of injury, leukocytic infiltrate, or endothelial cell activation up to 70 days postoperatively, despite deposition of murine complement. Host reconstitution with human peripheral blood mononuclear cells resulted in a discrete population of circulating T cells that did not infiltrate or injure the grafts up to 28 days after adoptive transfer. Administration of porcine interferon-gamma for up to 28 days sustained the expression of graft vascular cell adhesion molecule-1 and major histocompatibility complex antigens, but did not initiate recruitment of human leukocytes. In contrast, treatment with human tumor necrosis factor for 7 days induced the de novo expression of porcine E-selectin by graft endothelial cells and elicited human T cell infiltration and human peripheral blood mononuclear cell-dependent vascular injury. CONCLUSIONS: The human peripheral blood mononuclear cell-severe combined immunodeficiency/beige mouse model identifies a significant difference between human T cell allogeneic and xenogeneic responses in vivo. Xenografts with quiescent endothelium are not infiltrated or injured by T cells under the same conditions in which allografts are rejected. Activation of pig coronary artery endothelial cells by human tumor necrosis factor, but not porcine interferon-gamma, elicits cellular xenoresponses.

BXMAS1 identifies a cluster of homologous genes differentially expressed in B cells.

Characterization of genes activated by anti-IgM crosslinking of BL2 cells identified one gene, designated BXMAS1, that is predicted to be a novel cell surface receptor. The time course of activation indicates maximal transcriptional induction after 24 h. The predicted protein contains 977 aa residues, with a cytoplasmic domain containing 2 ITIM motifs. The ectodomain of the protein contains 6 repeats of characteristic 93 aa sequences which we have designated BXMAS1 domains. These domains correspond to 6 out of 8 Ig-like domains in BXMAS1. A search of the human genome revealed 5 additional closely linked homologous genes many of which contain BXMAS1 domains as well. Analysis of expression in cell lines and tissues suggests a general restriction of expression of these genes to B cells. These genes may be involved in B cell development and differentiation in peripheral lymphoid organs and may be useful markers of B cell stages.

Defining the specific physiological requirements for c-Myc in T cell development.

c-Myc is associated with cell growth and cycling in many tissues and its deregulated expression is causally implicated in cancer, particularly lymphomagenesis. However, the contribution of c-Myc to lymphocyte development is unresolved. We show here that the formation of normal lymphocytes by c-Myc-/- cells is selectively defective. c-Myc-/- cells are inefficient, in an age-dependent manner, at populating the thymus, and subsequent thymocyte maturation is ineffective: they fail to grow and proliferate normally at the late double-negative (DN) CD4-CD8- stage. Because N-Myc expression in thymocytes usually declines at the late DN stage, these results confirm that the nonredundant contributions of Myc family members to development are related to their distinct patterns of developmental gene expression.

Altered gene expression upon BCR cross-linking in Burkitt's lymphoma B cell line.

Burkitt's lymphoma cell line, BL2 was stimulated by surface BCR cross-linking and altered gene expression was analyzed by RDA methodology. Consistent with previous reports, we detected up-regulated MDC, IL6R and adhesion molecule LFA1. We also detected gene expression of SIRPalpha, anti-apoptotic A-20, signal regulatory SLP76 and BCAR3, DNA binding proteins EGR2 and DEC1 in addition to some new genes.

In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2- but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.

Tumor cells expressing membrane-bound form of IL-4 induce antitumor immunity.

Local cytokine concentrations are required for inhibition of tumor growth with less toxic side-effects. However, genetically engineered tumor cells secreting cytokines still induce toxicity and activate bystander cells. To circumvent such problems, membrane-bound forms of IL-4 (IL-4m) were expressed on MethA fibrosarcoma tumor cells. Chimeric forms of IL-4 with the type I transmembrane protein CD4 or type II transmembrane protein TNF were designed to express IL-4 in opposite orientations on the tumor cell surface. The IL-4m on tumor clones was able to support cell growth of the IL-4 dependent cytotoxic cell line (CT.4S) and the Th2 cell clone (D10). Furthermore, the IL-4m tumor clones stimulated proliferation of 2C TCR transgenic spleen cells which are responsive to Ld MHC class I molecules. Expression of the IL-4/TNF chimeric protein on MethA cells elicited antitumor immunity and protected from MethA tumor challenge. The proposed tumor vaccine may serve as an effective gene therapy method to avoid the toxicity of recombinant cytokines and bulk bystander leukocyte stimulation encountered in conventional cytokine gene therapy.

Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression.

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.

Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses.

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-kappaB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

Characterization of the human antiporcine immune response: a prerequisite to xenotransplantation.

Successful xenotransplantation necessitates solving problems of hyperacute rejection and understanding the cellular immune responses that occur. Considerable progress has been made in our understanding of the molecular genetic basis of the rapid hyperacute antibody-mediated rejection mechanisms that occur in xenogeneic organ rejection. In parallel, strategies involving the use of transgenic animals expressing complement inhibitors are beginning to offer encouraging evidence that hyperacute rejection can be overcome. A greater understanding of cell-mediated immune interactions is now required to achieve long-term xenograft survival. Current studies are focused on T cell receptor (TCR)/major histocompatibility complex (MHC) and costimulatory signals that activate human CD4 and CD8 T cells.

Complex regulation of Ly-6E gene transcription in T cells by IFNs.

The complexity of IFN-mediated regulation of the murine Ly-6E gene in T cell lines is highlighted by the following observations: 1) multiple regulatory regions are present within different parts of the Ly-6E promoter and are necessary for IFN inducibility of the Ly-6E gene, 2) multiple transcription factors including Oct-1 and Oct-2 and the high mobility group (HMG) protein HMGI(Y) bind to regulatory elements present within the G region required for both IFN-alphabeta and IFN-gamma responses, 3) mutational analysis of the G region reveals that a complex interaction exists between the factors binding to this region as shown by their mutual interdependence for detection in DMSA, and 4) inhibition of expression of HMG proteins by antisense HMGI-C RNA in EL4 cells causes the loss of IFN-alphabeta and IFN-gamma inducibility of the endogenous Ly-6 gene. These findings taken together suggest that, in response to IFN treatment, an HMG protein-dependent complex involving multiple regulatory factors is assembled and is required for IFN inducibility of the Ly-6E gene.

Dominant negative suppression of major histocompatibility complex genes occurs in trophoblasts.

BACKGROUND: Polymorphic class I and II major histo-: compatibility complex (MHC) genes are not transcribed in trophoblasts although many immune system cells express these genes constitutively. To study the molecular biology of MHC suppression for the purposes of potential transgenic animal development, we examined the effect on MHC expression in B cells by fusing them with trophoblasts. METHODS: Trophoblasts and B cells with separate selection markers were fused with polyethylene glycol. After growth in double selection media, the hybrids were analyzed for HLA-A, -B, -C, -DR, -DP, and -DQ expression by fluorescence-activated cell scanning and class I and II mRNA by Northern blotting. Class II promoter activity in trophoblasts was then analyzed by transfection of a lethal reporter construct and subsequently, the class II transactivator. RESULTS: Class I and II surface antigens and their corresponding mRNA were completely suppressed in the hybrids. The lethal reporter construct demonstrated that class II suppression resulted from lack of activation of the class II promoter. This in turn was caused by lack of functional class II transactivator. CONCLUSIONS: These data indicate that dominant negative trophoblast factors, either directly or indirectly, suppress expression of the MHC genes. If these factors can be cloned, the potential exists for developing transgenic animals that cannot express MHC or peptide antigen to T cell receptors through the MHC system.

STAT5 interaction with the T cell receptor complex and stimulation of T cell proliferation.

The role of STAT (signal transducer and activator of transcription) proteins in T cell receptor (TCR) signaling was analyzed. STAT5 became immediately and transiently phosphorylated on tyrosine 694 in response to TCR stimulation. Expression of the protein tyrosine kinase Lck, a key signaling protein in the TCR complex, activated DNA binding of transfected STAT5A and STAT5B to specific STAT inducible elements. The role of Lck in STAT5 activation was confirmed in a Lck-deficient T cell line in which the activation of STAT5 by TCR stimulation was abolished. Expression of Lck induced specific interaction of STAT5 with the subunits of the TCR, indicating that STAT5 may be directly involved in TCR signaling. Stimulation of T cell clones and primary T cell lines also induced the association of STAT5 with the TCR complex. Inhibition of STAT5 function by expression of a dominant negative mutant STAT5 reduced antigen-stimulated proliferation of T cells. Thus, TCR stimulation appears to directly activate STAT5, which may participate in the regulation of gene transcription and T cell proliferation during immunological responses.

Structure/function characterization of porcine CD59: expression, chromosomal mapping, complement-inhibition, and costimulatory activity.

BACKGROUND: Complement regulatory proteins have become important targets to potentially modulate inflammatory reactions or transplant rejection. Since pig into human xenotransplantation could potentially overcome the enormous shortage of donor organs and tissues, characterization of porcine complement regulatory proteins is critical. METHODS: The porcine CD59 cDNA has been isolated from porcine aortic endothelial cells and its structure determined. In addition, a molecular genetic analysis of the gene and its transcriptional properties and a functional analysis have been performed utilizing the transfected cDNA. RESULTS: The most prominent mRNA species is 1.8 kilobases but cloned reverse transcriptase polymerase chain reaction products suggest that multiple polyadenylation sites are utilized. Gene mapping was performed utilizing a polymorphism identified in the 3' UT, and the gene was localized to within 3 cM of follicle-stimulating hormone, beta polypeptide in the middle of the chromosome 2 linkage map. RNA expression was equivalent in endothelial, kidney, and testis cell lines. Comparisons have been made with CD59 sequences from other species to identify possible important domains of the protein. The cDNA has been utilized to express an epitope-tagged or wild-type protein either transiently on COS-7 cells or stably in Chinese hamster ovary cells. The porcine CD59 protein effectively inhibited the antibody-mediated lytic activity of both porcine and human complement. In contrast to human CD59, porcine CD59 is incapable of providing costimulation to human T cells. CONCLUSIONS: These data suggest that overexpression of porcine CD59 might be more effective than human CD59 in prolonging xenograft survival with transgenic pig organs because of reduced immunoreactivity.

Cell-cycle arrest and apoptosis hypersusceptibility as a consequence of Lck deficiency in nontransformed T lymphocytes.

By using antisense RNA, Lck-deficient transfectants of a T helper 2 (Th2) clone have been derived and shown to have a qualitative defect in the T cell receptor signaling pathway. A striking feature observed only in Lck-deficient T cells was the presence of a constitutively tyrosine-phosphorylated 32-kDa protein. In the present study, we provide evidence that this aberrantly hyperphosphorylated protein is p34(cdc2) (cdc2) a key regulator of cell-cycle progression. Lck-deficient transfectants expressed high levels of cdc2 protein and its regulatory units, cyclins A and B. The majority of cdc2, however, was tyrosine-phosphorylated and therefore enzymatically inactive. The transfectants were significantly larger than the parental cells and contained 4N DNA. These results establish that a deficiency in Lck leads to a cell-cycle arrest in G2. Moreover, transfected cells were hypersusceptible to apoptosis when activated through the T cell receptor. Importantly, however, this hypersusceptibility was largely reversed in the presence of T cell growth factors. These findings provide evidence that, in mature T lymphocytes, cell-cycle progression through the G2-M check point requires expression of the Src-family protein tyrosine kinase, Lck. This requirement is Lck-specific; it is observed under conditions in which the closely related Fyn kinase is expressed normally, evincing against a redundancy of function between these two kinases.

Distinct regulatory mechanisms for interferon-alpha/beta (IFN-alpha/beta)- and IFN-gamma-mediated induction of Ly-6E gene in B cells.

The murine Ly6-E gene is transcriptionally induced by interferon-alpha/beta (IFN-alpha/beta) and IFN-gamma in a variety of distinct cell types. The mechanism of IFN inducibility in B-cell lines was investigated by deletion analysis of the promoter and by identifying DNA binding proteins in mobility shift assays. A region located in the distal part of the promoter at -2.3 kb contributed to inducibility by both types of IFNs. This region contains a novel element in addition to the previously well-characterized IFN-stimulated response element (ISRE). The probes containing ISRE detected IFN-inducible complexes in mobility shift assays and the signal transducer and activator of transcripition-1 was found to be in these complexes from cells treated with either type of IFN. An additional element present in the proximal part of the promoter at position -109 is also required for IFN-alpha/beta-mediated induction. These data suggested a cooperative interaction between these physically disparate regulatory regions. A crucial role for HMGI(Y) protein in this cooperative multiprotein complex is supported by the evidence that inhibition of HMGI(Y) expression via antisense RNA results in the loss of IFN-alpha/beta-mediated induction of the Ly6-E gene. These results show the complexity involved in achieving cell-type specificity in IFN-mediated gene regulation.