Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.     

Involvement of TNF-like weak inducer of apoptosis in the pathogenesis of collagen-induced arthritis

TNF-like weak inducer of apoptosis (TWEAK) is a type II membrane protein belonging to the TNF family that regulates apoptotic cell death, cellular proliferation, angiogenesis, and inflammation. However, the role of TWEAK in the pathogenesis of rheumatoid arthritis (RA) remains unclear. In this study, we have investigated the effect of neutralizing anti-TWEAK mAb on the development of collagen-induced arthritis (CIA), a well-established murine model of RA. Administration of anti-TWEAK mAb significantly ameliorated paw swelling, synovial hyperplasia, and infiltration of inflammatory cells. The levels of proinflammatory chemokines such as MCP-1 and MIP-2 in serum and knee joints were reduced by this treatment. Consistently, recombinant TWEAK enhanced the proliferation of MCP-1 and MIP-2 production by synovial cells from CIA mice in vitro. Histological examination also revealed that the treatment with anti-TWEAK mAb suppressed the development of small vessels in synovial tissues. These results indicated anti-inflammatory and antiangiogenic effects of the TWEAK blockade in CIA, which may be also beneficial for the treatment of RA.     

Critical role of CD11a (LFA-1) in therapeutic efficacy of systemically transferred antitumor effector T cells

The systemic adoptive transfer of activated T cells, derived from tumor-draining lymph nodes (LNs), mediates the regression of established tumors. In this study, the requirement of cell adhesion molecules, CD11a/CD18 (LFA-1), CD54 (ICAM-1), CD49d/CD29 (VLA-4), and CD106 (VCAM-1), for T cell infiltration into tumors and antitumor function was investigated. Administration of anti-CD11a mAb completely abrogated the efficacy of adoptive immunotherapy for both intracranial and pulmonary metastatic MCA 205 fibrosarcomas. In contrast, adoptive immunotherapy was effective in animals treated with anti-CD49d mAb, anti-CD106 mAb, anti-CD54 mAb, or in CD54 knockout recipients. Trafficking of transferred cells to the intracranial tumor was not affected by any of the mAb. However, the tumor-specific secretion of IFN-gamma by activated LN T cells was suppressed by anti-CD11a mAb or anti-CD54 mAb. To account for the different effects of CD11a and CD54 blockade in vivo, an additional CD11a/CD18 ligand, CD102 (ICAM-2), was demonstrated on tumor-associated macrophages but not on tumor cells. These results show that CD11a mediates a critical function in interactions between effector T cells, tumor cells, and host accessory cells in situ leading to tumor regression.     

Suppression of chronic antigen-induced arthritis in rats by a monoclonal antibody against the T cell receptor alpha beta.

We have investigated a role for T cells in chronic antigen-induced arthritis in rats employing a monoclonal antibody (R73 mAb) against the T cell receptor alpha beta. Treatment with R73 mAb from the time of intra-articular antigenic challenge blocked completely the induction of chronic, but not acute ovalbumin-induced arthritis in sensitized rats. Histologically, treatment-controlled arthritic rats exhibited marked hyperplasia of synovial membrane with pronounced infiltration of inflammatory cells including alpha beta + T cells in the chronic phase of arthritis. In contrast, R73 mAb-treated rats had almost normal joint histology. Treatment with R73 mAb after onset of arthritis was also effective in suppressing the progression of chronic antigen-induced joint inflammation. The preventive and suppressive effects of the mAb on chronic antigen-induced arthritis were associated with marked depletion of alpha beta + T cells in peripheral blood. The DTH but not the humoral response to ovalbumin in sensitized rats was suppressed significantly by R73 mAb. Thus, alpha beta + T cells appear to have a central role in both induction and progression of chronic antigen-induced arthritis.     

Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.

We propose that leukocyte-derived cytokines induce the expression of adhesion molecules on the surface of neural cells that facilitates the subsequent attachment of leukocytes. Leukocyte adherence may contribute to some of the neural cell injury seen with various inflammatory diseases of the nervous system. With an in vitro model system, we have shown that mononuclear leukocytes bind to human neuroblastoma and cortical neuron cells only after the neural cells are stimulated with TNF-alpha. TNF-alpha stimulates expression of vascular cell adhesion molecule-1 (VCAM-1) in both of these neural cell lines. VCAM-1 mRNA is increased and VCAM-1 protein can be identified on the neural cell membranes with a new VCAM-1-specific mAb, CL40/2 F8. TNF-alpha also induces ICAM-1 in both of these neural cell lines. Leukocyte beta 1 (CD29) and beta 2 (CD18) integrins and their respective ligands, ICAM-1 and VCAM-1, on neural cells appear to be the dominant ligands mediating MNL:neural cell adhesive interactions. mAb to CD18 block 32 to 57% of the MNL binding to neural cells; similar inhibition is seen with mAb to ICAM-1. mAb to CD29 block 16 to 17% of the MNL binding to the neural cells suggesting that leukocyte beta 1 integrins and neural VCAM-1 may be a second route for MNL:neural cell interactions. Addition of both anti-CD18 and anti-CD29 mAb have an additive blocking effect; both ligand pairs may participate in MNL adhesion to neural cells, reminiscent of the multiplicity of ligands used by MNL when binding to endothelium.     

Differential role of distinct determinants of intercellular adhesion molecule-1 in immunologic phenomena

This study analyzed 1) the relationship between the molecules recognized by anti-intercellular adhesion molecule-1 (ICAM-1) mAb RR 1/1 and by anti-96K melanoma-associated Ag mAb CL203.4 in lymphoid cells, 2) the induction of ICAM-1 on activated PBMC, and 3) the functional activity of distinct and spatially distant determinants recognized by mAb CL203.4 and RR1/1. Sequential immunoprecipitation experiments showed that the determinant recognized by mAb CL203.4 is expressed on a slightly broader population of ICAM-1 molecules than that defined by mAb RR1/1. Serologic and immunochemical assays have shown that ICAM-1 is induced on lymphocytes activated with Con A, PHA-M, IL-2, allogeneic HLA mismatched lymphocytes and autologous PHA-M-activated T cells. However, ICAM-1 was not detected on lymphocytes incubated with IFN-gamma. Incubation of monocytes with LPS induced ICAM-1 in the subpopulation which lacks it and increased its density on the cells which express it. Induction of ICAM-1 is an early event in the activation process and precedes the appearance of IL-2 and transferrin receptors. Comparison of the functional activity of the anti-ICAM-1 mAb CL203.4 and RR1/1 showed that both of them inhibit to a similar extent proliferation of lymphocytes stimulated with PHA-M and with allogeneic lymphocytes, but that only mAb RR1/1 inhibits PMA-induced aggregation of cultured B lymphoid cells JY, of promonocytic cells U-937 and of PHA-blasts as well as LAK cell-mediated cytotoxicity of target cells. mAb CL203.4 represents the first example of anti-ICAM-1 mAb without inhibitory effect on the aggregation of lymphoid cells. The differential functional activity of mAb CL203.4 and RR1/1 does not reflect differences in their affinity, because they display a similar affinity constant to lymphoid cells. These results suggest that distinct determinants of ICAM-1 play a different role in immunologic phenomena.     

Blockade of IFN-gamma does not affect the arthritogenicity of T cells generated during the induction of adjuvant arthritis but exacerbates the polyarthritis produced by adoptive transfer of arthritogenic effector cells

IFN-gamma production is prominent in some models of autoimmune disease, including adjuvant arthritis (AA), but the role of IFN-gamma in the pathogenesis of these diseases is uncertain. Experimental manipulation (administration of cytokine, blocking cytokine action with specific antibody, disruption of genes encoding the cytokine or its receptor) has revealed both pro- and anti-inflammatory effects, depending on the nature of the manipulation and the timing of the treatment. We examined separately the effects of cytokine blockade during the afferent and efferent phases of AA in Dark Agouti rats, using an adoptive transfer system. Effects of IFN-gamma on the efferent phase were investigated by treating recipients with mAb DB-1, which blocks the activity of rat IFN-gamma. When treatment was commenced before cell transfer, the resulting polyarthritis was more severe than in controls treated with normal IgG. Commencing treatment after the adoptively transferred disease had become established caused neither amelioration nor exacerbation, but did cause some delay in resolution. In contrast, treatment of donors did not appear to affect the generation of arthritogenic cells. The main effect of IFN-gamma appears to be modulation of the arthritogenicity of the migratory effector T cells that can transfer AA.     

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.     

The effect of anti-adhesion molecule antibody on the development of collagen-induced arthritis.

In order to study how inflammatory cells including autoimmune lymphocytes interact with each other to develop collagen-induced arthritis (CIA), we injected monoclonal antibodies against mouse LFA-1 and ICAM-1 into DBA/1 mice immunized with type II collagen (CII). Both antibodies suppressed the development of CIA. These antibodies showed no effect on anti-CII antibody response, although they both significantly suppressed DTH response. It was suggested that anti-adhesion molecule antibodies suppress CIA mainly through their effect on cell-mediated immunity, without affecting humoral immunity under the conditions used.     

Ligation of ICAM-1 molecules inhibits target cell-induced granule exocytosis of IL-12-activated natural killer cells

The importance of cell adhesion molecules such as ICAM-1 is emphasized in cell-to-cell interactions that are critical in the generation of effective immune reactions. In this study, the involvement of ICAM-1 in natural killer (NK) cell activities was characterized in IL-12-activated human NK cells. To address the question of whether ligation of ICAM-1 molecules can modulate NK cell cytolytic activities, a 4-h (51)Cr-release assay was performed after pretreatment of NK cells with R6.5 mAb (anti-human ICAM-1 mAb). Ligation of membrane ICAM-1 molecules significantly inhibited IL-12-enhanced NK cytotoxicity against K562, and the pretreatment of neutralizing soluble ICAM-1 with R6.5 mAb blocked this inhibitory effect. The involvement of Ca(2+)-dependent granular exocytosis was evaluated. BLT esterase assay demonstrated that the ligation of ICAM-1 molecules inhibited granular exocytosis of NK cells. Additionally, the ICAM-1-mediated inhibition of Ca(2+) flux in NK cells was detected using Fluo-3AM, while the pretreatment of NK cells with R6.5 mAb did not affect conjugate formation between NK and K562 cells. Collectively, these results suggest that the signals transduced from ICAM-1 molecules might be sufficient to induce inhibitory effects on NK cells.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

Role of T lymphocytes in adjuvant arthritis. II. Different subpopulations of T lymphocytes functioning in the development of the disease.

The effects of treatments with cyclophosphamide (CY), hydrocortisone and anti-thymocyte sera (ATS) on the development of adjuvant arthritis (AA) were examined in WKA rats inoculated with wax D to induce AA. A single injection of 25 to 50 mg/kg of CY given 2 to 3 days before wax D inoculation caused severe arthritis with high incidence, whereas larger doses of CY were less efficient. Furthermore, the enhancing effect of CY pretreatment was abolished by passively transferred normal syngeneic thymocyreated with ATS and guinea pig C. On the basis of these results and the previous observations on the effect of adult thymectomy and low-dose irradiation, we concluded that this enhancing effect of CY pretreatment was caused by selective depletion of suppressor T lymphocytes. Pretreatment with 12.5 mg of hydrocortisone also caused severe arthritis. This enhancing effect of hydrocortisone could also be due to the elimination of those suppressor cells. In contrast, in vivo pretreatment with ATS showed striking inhibition on the development of AA, suggesting that ATS could eliminate T lymphocytes that were responsible for eliciting this disease. Thus it appears that at least two T cell subpopulations are involved in the development of AA, one is an ATS-sensitive T2 subpopulation that is effective for induction of AA and the other is a T1 subpopulation that regulates this disease process.     

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase

Intercellular adhesion molecule (ICAM)-3, a recently described counter- receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1- mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.     

CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1

CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral over-expression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process.     

Heterogeneous effects of IFN-gamma in adjuvant arthritis

In an attempt to evaluate the role of IFN-gamma in autoimmune arthritis, we tested the effects of IFN-gamma and anti-IFN-gamma mAb (DB-1) in various phases of arthritis development in a rat model for rheumatoid arthritis; the adjuvant arthritis (AA) model, induced by immunization with CFA. In addition, the effects of IFN-gamma were tested in vitro on T cell clones derived from rats afflicted with AA. T cell clone A2b, which has been shown to be arthritogenic secreted low amounts of IFN-gamma and its Ag-specific proliferation was inhibited by IFN-gamma. In contrast, clone A2c, which can inhibit the development of AA, produced high amounts of IFN-gamma and its proliferation was increased by IFN-gamma. In vivo administration of IFN-gamma 24 h before CFA caused an enhancement of arthritis, whereas giving IFN-gamma 24 to 48 h after CFA suppressed the disease. Administration of IFN-gamma between day +4 to +12 or between day +12 to +24 increased the severity of the first phase of the disease, but had no effect later. Administration of DB-1 1 to 2 days before adjuvant or between day +4 to +8 substantially decreased the disease, whereas DB-1 given from day +12 to +24 significantly enhanced it. Taken together, these results illustrate the heterogeneity of IFN-gamma in autoimmune arthritis and suggest a rational explanation for the possibly conflicting reports regarding the role(s) and effects of IFN-gamma in autoimmune processes. The multistage nature of T cell-mediated autoimmune arthritis may be due to the predominance of distinct T cell populations at different stages of the disease. The differences in the biologic activities of these T cells may be due to their patterns of lymphokine production.     

Metabolic fate of luteolin and its functional activity at focal site

Luteolin has been shown to possess potent antioxidant and anti-inflammatory/anti-allergic activities. In order to evaluate a chemopreventive role of luteolin in inflammatory responses involved in the pathogenesis of atherosclerosis and cancer etc., the metabolic fate of luteolin in rats and humans was investigated by HPLC analysis, and its effect on cell surface expression of adhesion molecules in human umbilical vein endothelial cells(HUVECs) was examined by ELISA. Luteolin monoglucuronide, which was a main metabolite, and free luteolin were detected in rat plasma and human serum. Luteolin monoglucuronide was hydrolyzed to free luteolin by beta-glucuronidase released from neutrophils stimulated with lonomycin and Cytocharasine B. Luteolin suppressed the TNF-alpha induced ICAM-1 expression significantly. Among nine flavonoids (40 microM) examined, chrysin, apigenine, quercetin and galangin also demonstrated suppressive effct on it. These results suggest the posssibility that deconjugation of luteolin monoglucuronide occurs and that free luteolin showed functional acyivities such as suppression of TNF-alpha induced ICAM- 1 expression at inflammation site.     

Activation of T cells recognizing an epitope of heat-shock protein 70 can protect against rat adjuvant arthritis.

We have previously reported that CD4+ T cells recognizing a peptide comprising residues 234-252 of the heat shock protein (HSP)70 of Mycobacterium tuberculosis (M.tb) in the context of RT1.B MHC class II molecule emerged in the peritoneal cavity during the course of Listeria monocytogenes infection in rats and suppressed the inflammatory responses against listerial infection via IL-10 production. We report in this work that pretreatment with peptide 234-252 of HSP70 derived from M.tb suppressed the development of adjuvant arthritis (AA) in Lewis rats induced using heat-killed M.tb. T cells from rats pretreated with peptide 234-252 produced a significant amount of IL-10 in response to the epitope. T cells from rats pretreated with the peptide and immunized with M.tb produced the larger amount of IL-10 in response to the peptide, but only a marginal level of IFN-gamma in response to purified protein derivative of M.tb. Administration of anti-IL-10 Ab partly inhibited the suppressive effect of pretreatment with peptide 234-252 on the development of AA. Furthermore, transfer of a T cell line specific for the epitope at the time of AA induction markedly suppressed AA. These findings suggested that T cells recognizing peptide 234-252 may play a regulatory role in inflammation during AA via the production of suppressive cytokines including IL-10.     

RhoA is involved in LFA-1 extension triggered by CXCL12 but not in a novel outside-in LFA-1 activation facilitated by CXCL9

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer's patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1-3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.     

LFA-1- and ICAM-1-dependent homotypic aggregation of human thymocytes induced by JL1 engagement

Cell-cell adhesion is essential for the appropriate immune response, differentiation, and migration of lymphocytes. This important physiological event is reflected in vitro by homotypic cell aggregation. We have previously reported that a 120 kDa cell surface glycoprotein, JL1, is a unique protein specifically expressed by immature double positive (DP) human thymocytes which are in the process of positive and negative selections through the interaction between thymocyte and antigen-presenting cells (APCs). The function of the JL1 molecule, however, is yet to be identified. We show here that anti-JL1 monoclonal antibody (mAb) induced the homotypic aggregation of human thymocytes in a temperature- and Mg2+-dependent manner. It required an intact cytoskeleton and the interaction between leucocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) since it was blocked by cytochalasin B and D, and mAb against LFA-1 and ICAM-1 which are known to be involved in the aggregation of thymocytes. Translocation of phosphatidylserine (PtdSer) through the cell membrane was not detected, implying that the molecular mechanism of JL-1-induced homotypic aggregation is different from that of CD99-induced homotypic aggregation. In summary, JL1 is a cell surface molecule that induces homotypic adhesion mediated by the LFA-1 and ICAM-1 interaction and cytoskeletal reorganization. These findings suggest that JL1 may be an important regulator of thymocyte development and thymocyte-APC interaction.