Organization of the human RH50A gene (RHAG) and evolution of base composition of the RH gene family

The alpha4 chain (CD49d), which constitutes one of the chains of alpha4beta1 (very late activating antigen-4 [VLA-4]) and alpha4beta7 integrins, mediates migration of T cells to extravascular spaces. The interaction between VLA-4 and vascular cell adhesion molecule-1 (VCAM-1) has been shown to be the critical pathway for the selective accumulation of eosinophils and basophils at sites of allergic inflammation. T lymphocytes are also specifically recruited into allergic sites, including the allergic asthmatic airway. Increased numbers of activated CD4+ cells expressing the DR antigen subset of the human leukocyte antigens (HLA-DR) appear in the allergic lung 48 h after allergen inhalation. The mechanisms by which these cells localize into the lung are still unknown. We report that stimulation of allergen-specific T cells with allergen in vitro resulted in enhanced expression of alpha4 chain (CD49d) as measured by receptor density on allergen-specific T-cell lines and T-cell clones. Kinetic studies showed that CD49d density was enhanced over a 24- to 48-h period in a time-dependent fashion, and was coordinately upregulated with HLA-DR expression. We also demonstrated that increased expression of CD49d on T-cell lines 24 h and 48 h after stimulation correlated with increased adhesion to the CS-1 fragment of fibronectin. In contrast, lymphocyte function-associated antigen-1b (LFA-1b) (CD11b), LFA-3 (CD58), and intercellular adhesion molecule-1 (ICAM-1) (CD54) expression did not change with allergen stimulation. We also showed that CD49d receptor density on T cells obtained by bronchoalveolar lavage (BAL) of allergic patients before and 48 h after allergen challenge was significantly higher than that on T cells taken from BAL of normal subjects and from controls with other inflammatory lung diseases. Taken together, these findings indicate that allergen stimulation activates allergen-specific T cells and coordinately induces increased CD49d receptor expression and binding to counterligands. We postulate that allergen-driven upregulation of CD49d, which together with the beta1 chain constitutes VLA-4 integrin, may be responsible for the selective accumulation of T cells in the allergic asthmatic lung.     

CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and delaying peripheral T cell deletion

In this report we show that activation of APC with an agonist anti-CD40 mAb profoundly alters the behavior of CD4 T cells in vivo. Stimulation of mice with anti-CD40 2 days before, but not 1 day after, administration of superantigen (SAg) enhanced CD4 and CD8 T cell clonal expansion by approximately threefold. Further, CD40 activation also delayed peripheral T cell deletion after activation. Dying, activated T cells were quantitated by detecting extracellular phosphatidylserine with concomitant staining for SAg-reactive T cells using a TCR Vbeta-specific mAb. Upon close examination, it was shown that CD40 activation delayed the death of the activated T cells. Additionally, it was found that enhanced survival of CD4 T cells was equally dependent on APC expression of B7-1 and B7-2. This is in contrast to CD8 T cells, which did not depend as much on B7-1 as B7-2. Thus, CD40 activation indirectly promotes T cell growth and delays the death of SAg-stimulated CD4 T cells in vivo. These data suggest that one way CD40 activation promotes a more robust immune response is by indirectly increasing the production of effector T cells and by keeping them alive for longer periods of time.     

The beta 1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers

Polymorphonuclear leucocyte (PMNL) accumulation in extravascular tissues and inflammatory exudates is dependent on their migration through blood vessel endothelium and then through connective tissue. Previously we utilized a barrier of human synovial and dermal fibroblasts (HSF or HDF) grown on microporous filters, as a model of PMNL migration through connective tissue. Those studies showed that beta 2 (CD18) and the beta 1 integrins, very late activation antigen-5 (VLA-5) and VLA-6, in part mediate this PMNL migration. Here we report that VLA-4, which can also be expressed at low levels on activated PMNL, is also involved in PMNL migration induced by C5a through fibroblast (HSF and HDF) barriers, because monoclonal antibody (mAb) to VLA-4 significantly inhibited (by 20-30%) PMNL migration. Blocking the function of CD18, VLA-5 or VLA-6 was not required for detection of the VLA-4-mediated migration. Combination treatment with mAb to VLA-4 and with mAb to VLA-5 or to VLA-6 further inhibited PMNL migration, irrespective of whether CD11/CD18 mechanisms were blocked with anti-CD18 mAb or not. Treatment of PMNL with a peptide based on the VLA-4-binding domain in the CS-1 fragment of fibronectin, but not a control peptide, inhibited PMNL migration to a comparable extent to treatment with mAb to VLA-4. A low level of VLA-4 was expressed on C5a-activated PMNL, detected by immunofluorescence flow cytometry. These results suggest that VLA-4 can be mobilized by human peripheral blood PMNL and can, in addition to VLA-5, VLA-6 and CD11/CD18 integrins, mediate PMNL migration through connective tissue. This is in marked contrast to PMNL transendothelial migration, where beta 1 integrins appear to play no significant role.     

CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis

The cyclic hexapeptide CWLDVC (TBC 772) is an antagonist of alpha4 integrins and a potent inhibitor of lymphocyte interactions with fibronectin, vascular cell adhesion molecule-1, and muscosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). As such, peptide TBC 772 effectively inhibits the activation of freshly isolated human T lymphocytes stimulated with purified vascular cell adhesion molecule-1 coimmobilized with anti-CD3 mAb. The influence of peptide binding on distinct sites of the alpha4beta1 complex was determined by flow cytometry and cellular adhesion assays employing a panel of mAbs. Binding of the alpha4-specific mAb L25 and the beta1-specific mAb 33B6 was not altered by the peptide; however, binding of mAb 19H8, which is specific for a combinatorial epitope of alpha4beta1, was dramatically inhibited. Treatment of lymphocytes with the peptide caused an increase in a ligand-induced epitope on beta1 integrin defined by mAb 15/7. In T cell activation studies using coimmobilized anti-CD3 mAb and the anti-integrin mAbs, the peptide had broader inhibitory activity, suppressing costimulation induced by all the integrin mAbs. The peptide was not generally toxic and was integrin selective in its suppressive activity, as coactivation by ligation of CD3 in conjunction with CD28 or CD26 was not affected. These results suggest that the antagonist peptide CWLDVC can effectively neutralize integrin coactivation systems by a mechanism independent of competitive binding.     

Semliki Forest virus infection leads to increased expression of adhesion molecules on splenic T-cells and on brain vascular endothelium

Semliki Forest virus A7 (SFV-A7) is a neurotropic alphavirus that leads to an asymptomatic encephalitis in adult immunocompetent mice. We studied the expression of leukocyte and endothelial cell adhesion molecules in the spleen and in the central nervous system (CNS) during SFV-A7 infection. Kinetics of the expression of LFA-1 alpha/CD11a, LFA-1 beta/CD18, Mac-1/CD11b, VLA-4/CD49d, ICAM-1/CD54 and L-selectin/CD62L was determined on splenic CD4+ and CD8+ T-cells and macrophages by flow cytometry. Time course of the expression of these antigens and VCAM-1/CD106 as well as viral antigens in the CNS was studied by immunoperoxidase staining. In the spleen, a sustained increase in LFA-1-expression and a temporary increase at day 7 in the expression of VLA-4, Mac-1 and ICAM-1 were detected on CD8+ T-cells. L-selection was down-regulated on CD4+ cells. Adhesion molecules on macrophages remained unchanged. In the CNS, expression of Mac-1+, VLA-4+ and LFA-1+ cells increased in parallel with the kinetics of the expression of their ligands ICAM-1 and VCAM-1 on brain vessels. Upregulation of adhesion of molecules peaked between days 5-8 and was most prominent in the cerebellar and brain stem white matter where viral antigens were most abundant. We conclude that the adhesion molecules profile of splenic T cells is altered during SFV-A7 infection which may influence their homing into the CNS. Macrophages are probably recruited non-specifically as a consequence of activation of the brain vascular endothelium in the inflamed areas of the brain.     

Interference of nonsteroidal antiinflammatory drugs with very late activation antigen 4/vascular cells adhesion molecule 1-mediated lymphocyte-endothelial cell adhesion

OBJECTIVE: To study the effect of nonsteroidal antiinflammatory drugs (NSAIDs) on the adhesion of peripheral blood lymphocytes (PBL) to activated human umbilical vein endothelial cells (HUVEC) under conditions that resemble blood flow. METHODS: Assays of adhesion of PBL to HUVEC or recombinant vascular cell adhesion molecule 1 (rVCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin were performed under continuous rotation at 37 degrees C. The phenotype of PBL subpopulations attached was characterized by flow cytometry. Lymphocytes were pretreated with different doses (5-100 microg/ml) of aceclofenac, diclofenac, indomethacin, or piroxicam or with inhibitory monoclonal antibodies (MAb) prior to the adhesion assays. The effect of NSAIDs on lymphocyte adhesion molecules was assessed by flow cytometry. To determine whether NSAIDs interfere with the affinity state of very late activation antigen 4 (VLA-4) integrin, we studied the effect of these drugs on the appearance of a beta1 activation-dependent epitope recognized by the HUTS21 MAb both on human T lymphoblasts and on synovial fluid lymphocytes (SFL). RESULTS: In the flow-resembling model, PBL-HUVEC adhesion was mainly mediated by the VLA-4/ VCAM-1 adhesion pathway. The major PBL subset attached was the CD3+, CD45RO+ memory T cell, with CD49d(high) expression. Aceclofenac, diclofenac, and indomethacin, but not piroxicam, were able to inhibit PBL adhesion to HUVEC or rVCAM-1. However, the quantitative expression of VLA-4 was not affected by treatment of PBL with any of the NSAIDs studied. On T lymphoblasts and SFL, mostly CD45RO+ cells, the expression of the beta1 activation-dependent epitope detected by HUTS21 MAb was significantly decreased by aceclofenac, diclofenac, and indomethacin. CONCLUSION: Some NSAIDs are able to inhibit the adhesion of PBL to HUVEC under conditions that resemble blood flow by interfering with the conformational change in VLA-4 that increases its affinity for VCAM-1.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.     

Expression of the CD8alpha beta-heterodimer on CD8(+) T lymphocytes in peripheral blood lymphocytes of human immunodeficiency virus- and human immunodeficiency virus+ individuals

CD8(+) T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8(+) peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8beta-chain expression on CD8(+) T lymphocytes and to clarify how its expression on CD8(+) T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8alpha beta-heterodimer, identifies CD8(+) T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8alpha antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8alpha beta staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8(+) T lymphocytes from HIV-1-infected individuals with the lowest CD4 counts showed the lowest levels of CD8alpha beta MF. To explore further this change in CD8alpha beta expression, we assessed the expression of 14 different cell surface molecules on CD8alpha beta+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8alpha beta staining was significantly reduced on CD8(+) T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8alpha beta expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28(-). Finally, we monitored the expression of the CD8alpha beta-heterodimer on PBL of eight HIV-1-infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8alpha beta-heterodimer. These results suggest that antibodies recognizing the CD8alpha beta-heterodimer are useful tools to specifically identify CD8(+) T lymphocytes. Moreover, the quantitative monitoring of CD8alpha beta expression allows the detection of discrete CD8(+) T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

CD11b+CD28-CD4+ human T cells: activation requirements and association with HLA-DR alleles

Engagement of CD28 induces a major costimulatory pathway required by many CD4+ T cells in addition to activation via the TCR. In the absence of signals provided by CD28, ligation of the TCR alone can induce anergy or apoptosis in CD28+ cells. However, we report here characterization of a distinct subset of CD4+ T cells that are CD28-. Three autoreactive CD4+ human T cell clones that could be activated to produce IL-2 and proliferate by anti-CD3 alone were found to lack expression of CD28. CD28- clones that were activated with anti-CD3 alone were not anergic to restimulation via CD3. The presence of CD28-CD4+ T cells was verified in peripheral blood, and their frequency ranged from 0% to >22% of CD4+ T cells in different individuals. The percentage of CD28-CD4+ T cells in the peripheral blood of 57 individuals was significantly correlated with specific class II MHC alleles. Persons with HLA-DRB1*0401 and DR1 alleles had significantly higher numbers of CD28- T cells, while individuals with HLA-DR2(15) had significantly fewer CD28-CD4+ T cells than the mean. Like the CD28- clones, CD28-CD4+ T cells isolated from peripheral blood proliferated upon CD3 cross-linking in the absence of costimulation. The finding that CD28-CD4+ T cells resist induction of anergy following engagement of the TCR in the absence of conventional costimulation demonstrates one mechanism by which autoreactive T cells can escape processes that censor self-reactivity. The MHC associations observed suggest a relationship with autoimmunity and loss of self-tolerance.     

Cell adhesion molecules--update

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.     

Increase in adhesion molecules on CD4+ cells and CD4+ cell subsets in synovial fluid from patients with rheumatoid arthritis

OBJECTIVE: To elucidate the role of adhesion molecules in the pathogenesis of rheumatoid arthritis (RA). METHODS: We evaluated their expression and that of an activation marker on CD4+ cell populations and CD4+ cell subsets in specimens of peripheral blood (PB) and synovial fluid (SF) obtained from 10 patients with RA and 7 with osteoarthritis (OA). A 2 or 3-color immunofluorescent method was used for analysis. RESULTS: The SF from both groups of patients showed a greater density of adhesion molecules including LFA-1 alpha, LFA-1 beta, CD2, VLA-4 alpha and VLA-5 alpha on CD4+ cells, and a higher percentage of CD4+HLA-DR+ cells compared with their PB. IN PB-CD4+ cell subsets from the arthritic and healthy subjects, the CD4+CD45RO+ cell population showed an increased expression of adhesion molecules compared with CD4+CD45RA+ cell population. The expression of adhesion molecules on circulating CD4+ cell population and CD4+ cell subsets from the patients with RA and OA was comparable to that from healthy subjects. SF from both groups of patients showed a higher percentage of CD4+CD45RO+ cells and a lower percentage of CD4+CD45RA+ cells. In SF-CD4+ cell subsets from patients with RA, the CD4+CD45RO+ cell population had an increased expression of VLA-4 alpha compared to the CD4+CD45RA+ cell population; however, there was no significant difference in other adhesion molecule expression and the percentage of HLA-DR+ cells between the 2 cell subsets. Furthermore, the expression of VLA-4 alpha on the CD4+CD45RO+ cell population in SF from patients with RA was significantly higher than that in matched PB. In CD4+CD45RA+ cell population from both groups of patients, SF showed an enhanced expression of adhesion molecules and an increased percentage of HLA-DR+ cells compared with matched PB. CONCLUSION: Our results suggest that increased expression of adhesion molecules and increased percentage of HLA-DR+ cells on CD4+ cells in SF may be responsible for cellular interactions between these cells and synovial cells or extracellular matrix.     

How tumor stage affects surgeons'' surveillance strategies after surgery for carcinoma of the upper aerodigestive tract

OBJECTIVE: To compare the expression of adhesion molecules on synovial T cells from patients with early spondyloarthropathy (SpA) and rheumatoid arthritis (RA), with special reference to the beta7 integrins alpha4beta7 and alphaEbeta7 in view of their intimate association with intestinal tissue. METHODS: Twenty-five synovial cell lines were generated by interleukin 2 (IL-2) expansion from synovial biopsies of patients with early SpA and RA, obtained from macroscopically inflamed synovial tissue by needle arthroscopy, and subsequently characterized by flow cytometry for CD3, CD4, CD8, L-selectin, CD11a, CD31, CD44, and alpha4beta7 and alphaEbeta7 integrin. RESULTS: In SpA, the beta7 integrin expression was increased, compared to RA. Furthermore, an inverse relation between alpha4beta7 and alphaEbeta7 was present in SpA (r = -0.75, p < 0.02), as on many mucosal T cells. In contrast, an opposite correlation was noted in RA (r = +0.84, p < 0.01), as similarly described on a subset of circulating T cells. CONCLUSION: Increased expression of beta7 integrins was noted on synovial T cell lines from SpA compared to RA, with discriminative correlations between alpha4beta7 and alphaEbeta7. This suggests a different origin of the synovial T cells in these diseases.     

Lymphotoxin alphabeta is expressed on recently activated naive and Th1-like CD4 cells but is down-regulated by IL-4 during Th2 differentiation

Lymphotoxin (LT) is a cytokine that orchestrates lymphoid neogenesis and formation of germinal center reactions. LT exists as a membrane heterotrimer of alpha and beta subunits and is secreted as a homotrimer, LTalpha3. Using LTbetaR.Fc, expression of LTalphabeta on CD4 T cell subsets was investigated in a TCR transgenic model. LTalphabeta was evident 24-72 h after activation of naive T cells with specific Ag, and declined thereafter. Early expression was independent of IFN-gamma and IL-12, however, IL-12 prolonged expression. LTalphabeta was reinduced within 2-4 h after Ag restimulation, but declined by 24 h regardless of IL-12 or IFN-gamma priming. Exposure of naive T cells to IL-4 did not affect early LTalphabeta expression at 24 h, but resulted in subsequent down-regulation. IL-4-differentiated Th2 effectors did not re-express LTalphabeta, and LTalphabeta was transiently found on Th1 clones but not Th2 clones. LTalpha3 and TNF were immunoprecipitated from supernatants and lysates of IL-12 primed cells but not IL-4 primed cells. These studies demonstrate that LTalphabeta is expressed by activated naive CD4 cells, unpolarized IL-2-secreting effectors, and Th1 effectors. In contrast, loss of surface LTalphabeta and a lack of LTalpha3 and TNF secretion is associated with prior exposure to IL-4 and a Th2 phenotype.     

Down-regulation of monocytic VLA-4 leads to a decreased adhesion to VCAM-1

The alpha 4 beta 1 integrin VLA-4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol-12-myristate-13-acetate (PMA) leads to a selective decrease in the VLA-4 alpha-chain expression, both at the RNA and protein level. Meanwhile the expression of beta 1 and that of alpha 5, another alpha-chain associating with beta 1, was seen to increase. The decrease of alpha 4 expression was restricted to monocytic cells, and was not observed on other VLA-4-positive cells tested (MOLT-4 T cells and HOS sarcoma cells). The down-regulation of the VLA-4 alpha-chain was followed by a decreased binding capacity of the cells to recombinant VCAM-1. This data indicates that while previous findings show that the integrin-dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.     

Expression and function of CTLA-4 in Th1 and Th2 cells

CTLA-4 is expressed on T cells after activation and shares homology with the CD28 costimulatory receptor. In contrast to CD28, CTLA-4 is thought to be a negative regulator of T cell activation. Cross-linking of CTLA-4 during activation of peripheral T cells reduces IL-2 production and arrests T cells in G1. Much less is known about the function of CTLA-4 in differentiated T cells. We have investigated the expression and function of CTLA-4 in established Th1 and Th2 clones and in bulk populations of Th1 and Th2 cells freshly derived in vitro from TCR transgenic splenocytes. We found that CTLA-4 was induced under similar conditions and with similar kinetics following activation of both Th1 and Th2 clones. However, CTLA-4 expression was much higher in Th2 than Th1 clones and lines. This was confirmed by flow cytometry, confocal microscopy, and Northern blot analysis. The ratio of surface to intracellular expression of CTLA-4 and its rate of endocytosis were similar in Th1 and Th2 clones. Inhibition of binding of CTLA-4 to its ligands using soluble anti-CTLA-4 mAb during stimulation with Ag increased the production not only of IL-2 by Th1 clones, but also that of IL-3 and IFN-gamma by Th1 clones and of IL-3, IL-4, IL-5, and IL-10 by Th2 clones. In contrast, when anti-CTLA-4 was coimmobilized with anti-CD3 and anti-CD28 mAbs, a decrease in the production of multiple cytokines was observed. We conclude that CTLA-4 can function to suppress the production of cytokines produced by both Th1 and Th2 cells.     

Expression of the VLA family of integrins in coeliac intestinal mucosa

The integrins, a family of cell surface proteins, mediate cell adhesion and may influence within the intestinal mucosa processes such as migration and/or proliferation and differentiation of enterocytes and lymphocytes. The aim of this study was to examine the distribution pattern of integrin subunits (VLA alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, beta1 chains) in normal intestinal mucosa and in that of patients with active coeliac disease (CD) and CD in remission. Immunohistochemical techniques and double immunostainings with monoclonal antibodies were used for investigation of the VLA alpha family of integrins and beta1 chain distribution. While the majority of the findings are consistent with the few data previously reported in the literature, surprising is the finding of a lack of expression of VLAalpha1 on the intraepithelial lymphocytes in the coeliac mucosa. The deficient VLA alpha1 expression on IEL in coeliac but not in normal mucosa may imply a genetic variation or a specific deficiency of gene expression during T cell differentiation and activation.     

Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.