Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.

Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.

The tetraspanin CD9 is preferentially expressed on the human CD4(+)CD45RA+ naive T cell population and is involved in T cell activation.

Human CD4+ T cells can be divided into reciprocal memory and naive T cell subsets based on their expression of CD45 isoforms and CD29/integrin beta1 subunit. To identify unique cell surface molecules on human T cells, we developed a new monoclonal antibody termed anti5H9. Binding of anti5H9 triggers a co-stimulatory response in human peripheral blood T cells. Retrovirus-mediated expression cloning has revealed that the antigen recognized by anti5H9 is identical to the tetraspanin CD9. We now show that human CD9 is preferentially expressed on the CD4(+)CD45RA+ naive T cell subset, and that CD9(+)CD45RA+ T cells respond preferentially to the recombinant beta2-glycoprotein I, compared to CD9-CD45RA+ T cells. Furthermore, anti5H9 inhibits both the recombinant beta2-glycoprotein I- and the recall antigen tetanus toxoid-specific T cell proliferation. These results suggest that the tetraspanin CD9 plays an important role in T cell activation.

Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines.

BACKGROUND: Cytokine imbalance and cellular migration to inflammatory sites are critical components of allergic diseases. Redirecting cytokine imbalance and inhibiting cell migration therefore represent important therapeutic strategies for the treatment of these disorders. OBJECTIVES: To study the in vitro effect of ebastine, a novel non-sedating H1 receptor antagonist, on cytokine secretion and migration of activated T cells, as well as production of pro-inflammatory cytokines by macrophages. METHODS: Peripheral T cells obtained from healthy volunteers were cultured in wells coated with the combination of anti-CD3 monoclonal antibody (mAb) and anti-CD26 mAb, anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb with PMA, in the presence or absence of ebastine. T cell proliferation and the production of cytokines were measured by [3H]thymidine incorporation assay and ELISA, respectively. In addition, transendothelial migration of T cells and production of pro-inflammatory cytokines by macrophages were examined. RESULTS: Ebastine inhibited T cell proliferation and the production of IL-4, IL-5, IL-6, and TNF-alpha by T cells under each co-stimulatory condition tested, whereas it exhibited no effect on the production of IL-2 or IFN-gamma. In addition, T cell migration and the production of such pro-inflammatory cytokines as TNF-alpha and IL-6 by macrophages were inhibited by ebastine. CONCLUSIONS: These results indicate that ebastine has a specific inhibitory effect on Th2-type cytokine production. Moreover, ebastine inhibited T cell migration and pro-inflammatory cytokine production by T cells and macrophages, suggesting that ebastine might be useful for the treatment of T cell-mediated allergic inflammatory disorders, including asthma, atopic dermatitis, and Th2-type autoimmune diseases.

Soluble CD26/dipeptidyl peptidase IV induces T cell proliferation through CD86 up-regulation on APCs.

CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of soluble CD26 (sCD26) resulted in enhanced proliferation of peripheral blood T lymphocytes induced by the recall Ag, tetanus toxoid (TT). However, the mechanism involved in this immune enhancement has not yet been elucidated. In this paper, we demonstrate that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of immune response. The cells directly affected by exogenously added sCD26 are the CD14-positive monocytes in the peripheral blood. Mannose-6 phosphate interfered with the uptake of sCD26 into monocytes, suggesting that mannose-6 phosphate/insulin-like growth factor II receptor plays a role in the transportation of sCD26 into monocytes. When sCD26 was added after Ag presentation had taken place, enhancement in TT-induced T cell proliferation was not observed. In addition, enhancement of TT-mediated T cell proliferation by sCD26 does not result from trimming of the MHC-bound peptide on the surface of monocytes. Importantly, we also showed that exogenously added sCD26 up-regulated the expression of the costimulatory molecule CD86 on monocytes through its dipeptidyl peptidase IV activity, and that this increased expression of CD86 was observed at both protein and mRNA level. Therefore, our findings suggest that sCD26 enhances T cell immune response to recall Ag via its direct effect on APCs.

Recognition of cell surface GD3 by monoclonal antibody anti-6C2 in rheumatoid arthritis synovial fluid: expression on human T cells with transendothelial migratory activity.

OBJECTIVE: We have previously reported that the anti-6C2 monoclonal antibody (mAb) defines a subset of human CD4+ memory T cells. The present study sought to determine the nature of the 6C2 molecule and the function associated with 6C2+ T cells, and to examine whether this T cell subset is involved in the pathophysiology of rheumatoid arthritis (RA). METHODS: Cytofluorographic analysis was performed for identification of T cell surface molecules displaying a distribution similar to that of the 6C2 molecule. T cells in the synovial fluid of RA patients were examined for expression of the 6C2 molecule. Transendothelial migratory activity was assessed by assay using monolayers of human endothelial cells. Specific reactivity of the anti-6C2 mAb was determined by immunoblotting on gangliosides separated by thin-layer chromatography, and flow cytometric analysis of the cells transfected with complementary DNA (cDNA) was performed for determination of the glycosyltransferases involved in biosynthesis of the gangliosides. RESULTS: On human peripheral T cells, the 6C2 molecule was distributed, by and large, in a pattern similar to that of CDw60, or O-acetyl-GD3. The majority (>70%) of synovial fluid T cells from patients with RA were found to be 6C2 positive, and those 6C2+ T cells exhibited a transendothelial migratory capacity that was inhibited by pretreatment of T cells with anti-6C2 mAb. Moreover, treatment of T cells with neuraminidase resulted in a loss of 6C2 expression as well as a reduction in the transendothelial migratory activity. Anti-6C2 mAb reacted specifically with GD3, but not with O-acetyl-GD3. The reactivity of anti-6C2 mAb was induced on the cell surface only by transfection with cDNA for GD3 synthase. CONCLUSION: The 6C2 molecule is a disialoganglioside, GD3, and is present on a subset of T cells with transendothelial migratory capacity. The 6C2/GD3 molecules, as well as 6C2/GD3+ T cells, appear to play a role in T cell migration and in the inflammation of RA.

Human herpesvirus type 8 and epstein-barr virus-associated cutaneous lymphoma taking anaplastic large cell morphology in a man with HIV infection.

Human herpesvirus type 8 (HHV-8, Kaposi's sarcoma-associated herpesvirus)-positive lymphoma taking anaplastic large cell morphology in the skin is described in a 46-year-old man with AIDS. Multiple erythematous nodules appeared on the trunk and extremities during the treatment of AIDS. Histological examination of cutaneous nodules showed dense infiltration of CD30 + atypical lymphoid cells in the deep dermis. Immunoglobulin JH gene rearrangement was detected in these lymphoma cells. Both Epstein-Barr virus-encoded small RNA and HHV-8 mRNA (T1.1/nut-1) were detected in these lymphoma cells by in situ hybridization. Remarkable retention of the pericardial fluid was observed at the same time that cutaneous lesions grew, and lymphoma cells in the pericardial fluid showed the same phenotype as the cutaneous lymphoma. Chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone effectively reduced both the cutaneous nodules and pericardial fluid. However, the patient died 4 months after diagnosis because of cytomegalovirus infection. As far as we know, this is the first report of an HHV-8-positive cutaneous lymphoma taking anaplastic large cell morphology. This case suggests the association of AIDS-related anaplastic large cell lymphoma with HHV-8.

Decreased dipeptidyl peptidase IV enzyme activity of plasma soluble CD26 and its inverse correlation with HIV-1 RNA in HIV-1 infected individuals.

Human plasma contains soluble CD26/dipeptidyl peptidase IV (sCD26/DPPIV) although its physiological significance remains unclear. To determine whether the plasma sCD26 levels have clinical relevance in HIV-1 infected individuals, the concentration and DPPIV enzyme activity of plasma sCD26 were measured. While there is no significant difference between the plasma levels of sCD26 in 90 HIV-1 infected individuals and in 79 uninfected controls, specific DPPIV enzyme activity of sCD26 was significantly decreased HIV-1 infected individuals (P < 0.0001). Specific DPPIV enzyme activity was correlated with the levels of CD4+ T cells (r = 0.247; P < 0.02), CD8+ T cells (r = 0.236; P < 0.03), and adenosine deaminase (r = 0.227; P < 0.05) and had an inverse correlation with HIV-1 RNA (Spearman's r = 0.474; P = 0.0012). Furthermore, recombinant sCD26 enhanced the in vitro PPD-induced response of lymphocytes from HIV-1 infected individuals with decreased specific DPPIV enzyme activity. These results suggest that the specific DPPIV enzyme activity of plasma sCD26 may contribute to the immunopathogenesis of HIV infection.

Human T lymphocyte populations which bind to P- or E-selectin are enriched with cells expressing core 2 O-glycans.

The present study was undertaken to investigate whether core 2 O-glycans are involved in binding of resting human T lymphocytes to P- or E-selectin and in recruitment of these cells to inflammatory sites. Freshly isolated human peripheral blood T lymphocytes were incubated with P- or E-selectin-coated dishes, and expression of core 2 O-glycans by the adherent and nonadherent cells was examined using the anti-1D4 mAb, which specifically recognizes human CD43 modified with core 2 O-glycans. The results indicated that both the P-selectin/adherent and E-selectin/adherent populations were significantly enriched with ID4+ cells, as compared with the initial population. An enrichment of ID4+ cells in the P- and E-selectin/adherent populations was observed in both CD4 and CD8 T cell subsets and even in the CD45RO+ memory CD4 T-cell subset. However, the anti-1D4 mAb did not inhibit binding of human T lymphocytes to P- or E-selectin, indicating that the 1D4 antigen itself is not directly involved in selectin binding. We also found that the percentage of ID4+ cells in synovial fluid T lymphocytes of rheumatoid arthritis patients was significantly increased as compared with normal peripheral blood T lymphocytes. Taken together, our results support the notion that core 2 O-glycans, which are located apart from the ID4 antigen, are involved in binding of human resting T lymphocytes to both P- and E-selectin, and these interactions may contribute to preferential recruitment of human memory CD4 T lymphocytes to inflammatory sites, including the synovium of rheumatoid arthritis patients.

CD26/dipeptidyl peptidase IV differentially regulates the chemotaxis of T cells and monocytes toward RANTES: possible mechanism for the switch from innate to acquired immune response.

CD26, a 110 kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) enzyme activity and plays an important role in T cell co-stimulation. In the present study, the function of CD26/DPPIV in transendothelial migration was examined using beta-chemokines as chemoattractants. When soluble recombinant CD26 (sCD26/DPPIV+) was added to the transendothelial chemotaxis system, chemotactic migration of T cells toward RANTES was significantly enhanced. Addition of sCD26 to 50 ng/ml of RANTES enhanced the migratory response by a factor of two compared to RANTES alone, whereas mutant soluble CD26 (mCD26), lacking the DPPIV enzyme activity, had no enhancing effect on RANTES-induced T cell migration. In the process of analyzing the mechanisms of the enhancement of T cell migration by sCD26, we showed that RANTES was cleaved by sCD26 under physiologic conditions at the precise site characteristic of its enzyme specificity. However, synthesized RANTES which lacks two N-terminal amino acids showed a chemotactic activity equivalent to full-length RANTES on T cells. Furthermore, addition of sCD26 showed enhancement of T cell migration induced by both forms of RANTES. In contrast to T cells, the truncated RANTES is inactive in chemotaxis of purified monocytes and supplement of sCD26 but not mCD26 reduced the migratory response of monocytes to RANTES. These results suggest that CD26/DPPIV differentially regulate the chemotactic response of T cells and monocytes to RANTES.

Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells.

Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1 D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4- T cells, implying that the 1 D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1 D4 was completely abolished when cells were treated with neuraminidase, while them remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.

Negative regulation of the anti-human immunodeficiency virus and chemotactic activity of human stromal cell-derived factor 1alpha by CD26/dipeptidyl peptidase IV.

Stromal cell-derived factor 1alpha (SDF-1alpha) is a chemokine that has been shown to prevent infection of T-tropic HIV strains and is a possible substrate of CD26/dipeptidyl peptidase IV (DPPIV). In this study, we show that SDF-1alpha was cleaved at the N-terminal region by CD26/DPPIV and as a result the inhibitory activity of SDF-1alpha against HIV infection disappeared. Moreover, the chemotactic activity of SDF-1alpha also disappeared specifically by DPPIV activity of recombinant soluble CD26. These results suggested that dissemination of T-tropic HIV strains in vivo may be facilitated by CD26/DPPIV via inactivation of functional SDF-1alpha.

Cytomegalovirus (CMV) retinitis and CMV antigenemia as a clue to impaired adrenocortical function in patients with AIDS.

OBJECTIVE: To elucidate the relationship between the activity of CMV disease and adrenocortical function in patients with AIDS. DESIGN AND PATIENTS: CMV retinitis and CMV antigenemia assay (CMV-Ag: numbers of polymorphonuclear leukocytes positive for CMV pp65 antigen per 1.5 x 10(5) cells) are the least invasive and easily accessible examinations to assess the CMV disease activity. All HIV-infected patients with CD4+ lymphocyte counts < 50 x 10(6)/l who were admitted to the Research Hospital of the Institute of the Medical Science (University of Tokyo) between May 1995 to April 1996 were included in this study. METHODS: Fundoscopic examination on CMV retinitis and CMV-Ag were chosen as methods to assess CMV activity because of their simplicity. Adrenocortical function was evaluated by basal plasma adrenocorticotropin, plasma cortisol, plasma aldosterone, plasma renin activity, and responses of plasma cortisol and plasma aldosterone to 250 micrograms intravenous cosyntropin [rapid adrenocorticotropin test (RAT)]. RESULTS: Thirty patients were enrolled in this study with a maximum CD4+ lymphocyte count of 32 x 10(6)/l. Eleven out of 30 patients showed impaired RAT response (37%). Fourteen out of 30 patients had CMV retinitis. A significant correlation was found between the presence of CMV retinitis and subnormal cortisol response (P < 0.005). Sixteen out of the 30 patients were CMV-Ag-positive. A significant correlation was found between CMV-Ag positivity and subnormal cortisol response to RAT (P < 0.005). CMV-Ag levels in the patients with subnormal cortisol response to RAT were significantly higher than those with normal response (P < 0.001). Importantly, five patients with subnormal cortisol response but not overt adrenal insufficiency at the time of RAT developed overt disease shortly afterwards. Autopsy was performed in six patients with subnormal cortisol response and showed multiple inclusion bodies indicative of CMV adrenitis. CONCLUSION: The adrenal gland is most frequently affected by CMV in AIDS patients. Our result suggests that CMV retinitis or CMV-Ag positivity independently serve as an indication of possible adrenal dysfunction.

[Clinical significance of anti-cardiolipin antibody in patients with systemic lupus erythematosus (SLE)].

Anti-cardiolipin antibody (aCL) in SLE patients reacts with cardiolipin/beta 2-GP1 complex. In order to disclose clinical significance of aCL in patients with SLE, aCL was determined by newly-developed anti-CL.beta 2-GP1 kit [Yamasa] EIA in 58 patients with SLE and the relationship between clinical manifestation of SLE and the presence of aCL was examined. Anti-cardiolipin antibody was positive in 20 patients (34.5%). In 20 SLE patients with positive aCL, livedo reticularis in 7, retinal vein thrombosis in 3, thrombophlebitis in 3 and other vasculo-occlusive episodes were observed as a characteristic clinical features of positive aCL. In contrast, a SLE patient complicated by ileal perforation due to necrotizing angiitis had negative aCL. Other clinical and laboratory features associated with aCL include recurrent fetal loss and thrombocytopenia.

Integrin VLA-5 negative primary plasma cell leukemia.

We present a case of primary plasma cell leukemia with Bence Jones proteinuria. After combination chemotherapy, leukemic cells and the urinary levels Bence Jones protein were decreased. Small lytic bone lesions were detected only in the skull. Typical plasma cells were rarely seen in peripheral blood on the hyperleukocytic phase, however they were increased in the advanced stages. The most important diagnostic sign was persistent expression of CD38 antigen on leukemic cells throughout the entire course of the illness and these leukemic cells expressed very late antigen-4 (VLA-4) but not VLA-5.

Differentiation of human lambda I variable regions with a monoclonal antibody to a defined, germ-line-encoded idiotope correlates with V lambda Ia chemical structure.

This article describes the characterization of a mouse monoclonal anti-idiotypic antibody (1H7B) prepared against a human monoclonal rheumatoid factor (RFSJ2) whose L chain utilized a V lambda I subgroup gene. The mAb 1H7B reacted with 6 of the tested 12 human V lambda I proteins, as well as with a newly produced lambda mAb whose V lambda gene usage has not as yet been determined. Because six of the 1H7B-positive mAb were heterogeneous with respect to both their VH gene utilization and antigenic specificity, and because mAb 1H7B did not react with any of the tested 43 kappa or 12 lambda proteins belonging to various subgroups other than V lambda I, mAb 1H7B appeared to be a V lambda I subgroup-specific reagent. The L chain specificity of mAb 1H7B was confirmed by Western blotting, and the inhibition of RFSJ2 binding to human Fc gamma by 1H7B provided additional evidence for the V region specificity of mAb 1H7B. The 11 sequenced V lambda I proteins used in this study were assigned to sub-subgroups by comparison with the previously published germ-line V lambda Ia, V lambda Ib, and V lambda Ic sequences. The mAb 1H7B only appeared to recognize V lambda Ia proteins as it reacted with five of the seven V lambda Ia, but not with three V lambda Ib or with one V lambda Ic protein. Because mAb 1H7B reacted with at least one V lambda Ia sequence in germ-line configuration, it appeared to be a marker for V lambda Ia sub-subgroup germ-line gene(s). The idiotope recognized by 1H7B was localized to the first framework region by inhibiting its binding to RFSJ2 with a synthetic peptide to the 11-24 amino acid region of V lambda Ia L chains. Comparison of the V lambda 1-24 region sequence of RFSJ2 with those of the two 1H7B-negative V lambda Ia mAb revealed a single amino acid difference at position 17, suggesting that the idiotope recognized by 1H7B encompassed this position.

The presence of anti-protein A antibodies in patients with rheumatoid arthritis.

Sixteen patients with rheumatoid arthritis (RA) were examined for the presence of anti-protein A antibodies. The F(ab')2 preparations from five RA patients showed significant binding to IgG-free protein A on ELISA. The protein A binding was further examined by immunoblotting. The F(ab')2 preparations of high protein A-binding protein gave a specific reaction with IgG-free protein A on nitrocellulose paper. This demonstrates the presence of anti-protein A antibodies in patients with RA. Those RA patients with anti-protein A antibodies had more active disease as judged by the Lansbury's activity index. The level of serum rheumatoid factor (RAHA) was significantly higher in patients with anti-protein A antibodies than in those without anti-protein A antibodies.

Thrombotic thrombocytopenic purpura in two patients with systemic lupus erythematosus: clinical significance of anti-platelet antibodies.

Thrombotic thrombocytopenic purpura (TTP) is a clinical syndrome of unknown etiology and has a high mortality rate due to disseminated platelet thrombi. However, the mechanism of platelet agglutination is not understood. Although an immunological mechanism has been suggested as the basis for the pathogenesis of TTP, any possible immune-mediated etiology remains unclear. The association of TTP with systemic lupus erythematosus (SLE) affords a unique opportunity to study such possibilities, because SLE is a prototype of autoimmune disease. This report describes two patients with SLE who developed TTP. The development of anti-platelet antibodies is one possible immunological mechanism for platelet agglutination in patients with SLE complicated by TTP. More importantly, patient J.Y., who had anti-platelet antibodies, responded dramatically to high doses of prednisolone.

CCA [N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt], a newly developed immunomodulating drug, suppresses T-cell activation by acting on macrophages.

The cellular mechanism of action of a newly developed drug, CCA, N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt, on PHA-, autologous mixed lymphocyte reaction (AMLR)-, and phorbol myristate acetate (PMA)-stimulated T-cell proliferation was investigated. Addition of 50 micrograms of CCA per milliliter suppressed PHA- and AMLR-stimulated T-cell proliferation. In contrast, CCA failed to suppress PMA-stimulated macrophage-depleted T-cell proliferation. After treatment of T cells or macrophages with CCA for 12 h, recombined T cells and macrophages were stimulated with phytohemagglutinin. [3H]Thymidine incorporation by T cells was suppressed when macrophages but not T cells were treated with CCA. These results indicate that CCA suppresses T-cell proliferation by acting on macrophages. The mechanism involved in this suppression of CCA was due to the loss of Ia antigen on macrophages and the loss of interleukin-1 (IL-1) secretion from macrophages.