Leukocyte Function-associated Antigen-1 Expression Research Articles

Differences in glomerular leukocyte infiltration between IgA nephropathy and membranoproliferative glomerulonephritis.

An important aspect in glomerular nephritic processes is the enhanced influx of leukocytes into the glomerulus. To investigate the mechanisms of intraglomerular leukocyte infiltration in IgA nephropathy (IgA-N) and membranoproliferative glomerulonephritis type I (MPGN-I), we immunohistochemically examined the intraglomerular expression of leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18), macrophage-1 (Mac-1, CD11b/CD18) and intercellular adhesion molecule-1 (ICAM-1, CD54) together with glomerular deposition of C3c and fibrinogen. In IgA-N (n=42), LFA-1+ cells were distributed mainly in glomeruli with intense expression of ICAM-1, and there was a positive correlation (P<0.001) between the number of LFA-1+ cells and the degree of ICAM-1 expression. Mac-1+ cells had no correlation with glomerular C3c deposition, but had a significant correlation with fibrinogen deposition (P<0.05). The number of LFA-1+ cells was significantly greater than of Mac-1+ cells (P<0.05). The number of LFA-1+ cells was strongly correlated with that of CD68+ cells (P<0.00001). In MPGN-I (n= 43), on the contrary, Mac-1+ cells correlated only with C3c deposition (P<0.001), and they were observed mainly in peripheral loops of glomerular capillaries where C3c was deposited with a similar distribution. However, there was no relationship between LFA-1+ cells and ICAM-1 expression. The number of Mac-1+ cells was greater than that of LFA-1+ cells (P<0.0001), and most Mac-1+ cells were identical to CD15+ cells. These results indicate the possibility that different mechanisms may cause glomerular leukocyte infiltration in various forms of human glomerulonephritis. The LFA-1/ICAM-1 pathway may play an important role in glomerular leukocyte infiltration in IgA-N, while the Mac-1/complement pathway may be important in MPGN-I. The former may promote mainly the infiltration of CD68+ cells, and the latter may promote that of CD15+ cells. In addition, Mac-1+ cells may act as fibrinogen and complement receptors in IgA-N and MPGN-I, respectively.

Leukocyte function-associated antigen 1-dependent adhesion of rat hepatoma AH66F cells and inhibition by protein kinase C inhibitors

When rat ascites hepatoma AH66F cells were incubated on a mesothelial cell (M-cell) layer for 1 hr, the adhesion rate of the cells to M-cells was ca. 46%. The protein kinase C (PKC) inhibitors, N-(2-methylpiperazyl)-5-isoquinolinesulfonamide (H-7) and N-ethoxycarbonyl-7-oxostaurosporine (NA-382), inhibited the adhesion of AH66F cells in a concentration-dependent manner, and the effect of NA-382 appeared after a treatment of more than 24 hr. The decreased adhesion rate after treatment with NA-382 for 48 hr was not further inhibited by addition of monoclonal antibodies of leukocyte function-associated antigen-1 (LFA-1) α- and β-chains and intercellular adhesion molecule-1 (ICAM-1) (WT.1, WT.3, and 1A29, respectively). The expression of LFA-1 α- and β-chains on the surface of the plasma membrane of AH66F cells was decreased after treatment with NA-382 for 48 hr; treatment with a potent inhibitor of cyclic AMP-dependent protein kinase, N-[2-( p-bromocinnamylamino)ethy l]-5-isoquinolinesulfonamide (H-89), did not affect the cell adhesion and the expression of LFA-1 molecules on AH66F cells. These results suggest that the expression of LFA-1 molecules on AH66F cells is regulated through the PKC pathway.

Expression of Activation-Related Molecules on Regional Lymph Node Lymphocytes in Human Lung Cancer

Using two- and three-color flow cytometry, we investigated the surface makers of regional lymph node lymphocytes (RLNL) in 54 patients with primary lung cancer in order to determine whether or not RLNL are in an activation state when compared with the corresponding peripheral blood lymphocytes (PBL) for the purpose of clarifying the characteristics of RLNL. RLNL showed a cell composition that was different from the corresponding PBL with a high proportion of CD3 + cells, CD4 + cells and CD20 + (B) cells as well as a low proportion of CD8 + cells and CD16 + (NK) cells. RLNL also contained a significantly higher proportion of CD45RO + T cells and a lower proportion of CD45RA + T cells in comparison to the corresponding PBL. Furthermore, we explored the activation-related molecules such as the interleukin-2 receptor (IL-2R) α chain, the IL-2R β chain, HLA-DR and leukocyte function-associated antigen 1 (LFA-1) on CD4 + and CD8 + cells. The data showed that the expression of CD45RO, the IL-2R α chain, HLA-DR on CD4 + cells, and those of CD45RO and HLA-DR on CD8 + cells were significantly higher in RLNL than in PBL. On the other hand, PBL showed a higher expression of the IL-2R β chain and LFA-1 only on CD8 + cells, which are thought to include CD8 + NK cells. When these activation-related molecules were analyzed on CD45RO + T cells, which are thought to be memory T cells, then the expression of the IL-2R α chain, HLA-DR on CD4 +CD45RO + cells and HLA-DR on CD8 +CD45RO + cells were significantly higher in RLNL than in PBL. Lastly, we analyzed the surface molecules according to such clinical factors as metastasis to the lymph nodes and the stage of lung cancer. Interestingly, RLNL with lymph node metastasis showed an increase in the percentage of CD20 + cells and a decrease in the percentage of CD4 + cells in comparison to those with benign lung disease, while these differences were not observed in PBL. The expression of CD45RO and LFA-1 on CD4+ and LFA-1 on CD8 + cells on RLNL in stage I + II is significantly higher in comparison with benign lung disease. Furthermore, RLNL in stage III + IV revealed the reduced expression of CD45RO, HLA-DR and LFA-1 on both CD4 + and CD8 + cells compared with those in stage I+II. These results thus demonstrated that RLNL were in a more activated state, particularly in stages I + II, than PBL. Whether these activation states in RLNL are directed against autologous tumor cells or not can hopefully be elucidated after the completion of further ongoing studies.

Induction of LFA-1 on Pluripotent CD34+ Bone Marrow Cells Does Not Affect Lineage Commitment

Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis remains obscure. Since LFA-1 is predominantly expressed by leukocytes, it is considered as a marker of late stage stem cell maturation when expressed on CD34+ bone marrow cells, and represents more mature hematopoietic progenitor cells. We observed that freshly isolated CD34+ bone marrow cells express LFA-1, and that the level of expression is highly variable. Interestingly, the expression of LFA-1 specific activation epitope L16 on these cells is low, even after culture. This demonstrates the LFA-1 is not activated, as was confirmed by low adhesion to ICAM-1. Culturing sorted CD34+ LFA-1+ cells in single cell per well assays in medium supplemented with SCF, Epo, IL-3, Il-6, GM-CSF, and G-CSF revealed that they gave rise to dispersed macrophage-like colonies, supporting the notion that CD34+LFA-1+ cells indeed consist of a mature committed cell population. In contrast, sorted CD34+LFA-1- cells had high proliferative potential and developed into large multilineage colonies within 14 days of culture. Unanticipated, in time course experiments we observed that these CD34+LFA-1- cells expressed LFA-1 within 24 hours upon culture. This induction was neither caused by the monoclonal antibody used to tag CD34 cells, nor dependent on growth factors present in the medium. These findings demonstrate that two populations of CD34+LFA-1+ cells can be discriminated: leukocyte lineage committed CD34+ cells in freshly isolated bone marrow cells, and multipotent CD34+ cells that acquired LFA-1 upon in vitro culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA-1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, providing those cells with adhesion receptors enabling migration in the periphery.

Colchicine interferes with L-selectin and leukocyte function-associated antigen-1 expression on human T lymphocytes and inhibits T cell activation.

Colchicine, which inhibits cell microtubule assembly by preventing polymerization of tubulin monomers, inhibits cell-mediated immune responses and promotes long-term survival of major histocompatibility complex-incompatible renal allografts in rats. Here we evaluated the effect of blocking cell microtubule assembly by colchicine on T cell and endothelial cell adhesion receptors involved in transducing signals for T cell activation. By using immunofluorescence flow cytometry analysis, evidence is presented that colchicine, in a dose-dependent fashion, downregulated L-selectin and leukocyte function-associated antigen-1, but not CD2 and CD44 on the surface of naive human peripheral blood lymphocytes. This effect was confirmed in two subsets of T lymphocytes, namely, CD45RA- and CD45RO-positive cells. However, colchicine did not influence the rapid shedding of L-selectin from T lymphocytes exposed to activating stimuli. Colchicine inhibited expression of interleukin-2 receptor on activated T lymphocytes. This effect was observed when T lymphocytes were stimulated with both anti-CD3 and anti L-selectin monoclonal antibodies. Colchicine also inhibited lymphocyte function in vitro as documented by inhibition of the human mixed lymphocyte response in a dose-dependent fashion. Moreover, colchicine downregulated surface expression of intercellular adhesion molecule-1 and E-selectin on activated human umbilical vein endothelial cells. These results indicate that blocking cell microfubule assembly inhibits surface expression of adhesion molecules on T cells and endothelial cells, and provides insights into the complex mechanisms of the action of colchicine in vivo.

Expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin and their ligands VLA-4 and LFA-1 in chronic venous leg ulcers

Background: Leukocyte binding to endothelial cells (ECs) is thought to contribute to the pathogenesis of leg ulcers caused by chronic venous insufficiency. In other systems, such binding is mediated by the interaction of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (on ECs), and leukocyte function-associated antigen-1 (LFA-1) and very late activated antigen-4 (VLA-4) (on leukocytes). Objective: Our purpose was to determine whether an increased expression of these adhesion molecules contributes to the pathogenesis of chronic venous insufficiency. Methods: Twenty-seven biopsy specimens of inflamed dermatoliposclerotic skin adjacent to venous leg ulcers were stained immunohistochemically with monoclonal antibodies against ICAM-1, VCAM-1, LFA-1, VLA-4, and E-selectin. Staining intensity was compared with that of normal skin. Results: Specimens of leg ulcers caused by chronic venous insufficiency showed increased expression of ICAM-1 and VCAM-1 but not of E-selectin on ECs. The expression of LFA-1 and VLA-4 on perivascular leukocytes was increased dramatically in comparison to healthy skin. Conclusion: Upregulation of ICAM-1 and VCAM-1 on ECs may contribute to the increased adherence and extravasation of LFA-1- and VLA-4-positive leukocytes in chronic venous insufficiency.

Regulation of the expression of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on a human eosinophilic leukemia cell line EoL-3

The effects of several cytokines and phorbol myristate acetate (PMA) on LFA-1 and ICAM-1 expression on a human eosinophilic leukemia cell line. EoL-3, were investigated and compared with those of a human monocytic leukemia cell line, U937. EoL-3 cells expressed large amounts of LFA-1 and small amounts of ICAM-1, and their expression was regulated similarly in EoL-3 cells and U937 cells. Interferon-γ (IFN-γ) enhanced ICAM-1 expression but not LFA-1 expression, and PMA augmented both LFA-1 and ICAM-1 expression. IFN-γ and PMA showed an additive effect on ICAM-1 expression. These results collectively suggest that expression of LFA-1 and ICAM-1 is regulated differently and that IFN-γ and PMA regulate the expression through different mechanisms. PMA but not IFN-γ induced homotypic adhesion of EoL-3 and U937 cells, suggesting that PMA but not IFN-γ activated the adhesive function of these cells. Staurosporin, an inhibitor of protein kinases (PKs), partly suppressed IFN-γ- and PMA-augmented expression of ICAM-1 on EoL-3 and U937 cells, but did not affect PMA-augmented LFA-1 expression, suggesting that staurosporin-sensitive PKs are involved in IFN-γ- and PMA-augmented ICAM-1 expression but not in PMA-augmented LFA-1 expression. The role of protein kinase C (PK-C) in these mechanisms was not revealed because a PK-C inhibitor, H-7, did not show any definitive effect on IFN-γ-and PMA-induced expression of LFA-1 and ICAM-1. Moreover, cyclic AMP (cAMP)- and cGMP-dependent pathways were not shown to be involved in the augmentation of the expression of these molecules.

Increased Expression of Leukocyte Function Associated Antigen-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1) by Alveolar Macrophages of Patients with Pulmonary Sarcoidosis

Leukocyte function associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) are cell adhesion molecules that play an important role in the capacity of monoculear phagocytes (MPs) to present antigens to T lymphocytes. Since in pulmonary sarcoidosis (PS) this capacity is increased at sites of disease activity, we studied the expression of LFA-1 and ICAM-1 on peripheral blood monocytes (BMs) and alveolar macrophages (AM) obtained by bronchoalveolar lavage (BAL) from normal subjects (n = 7) and patients with PS (n = 14). To accomplish this, immunocytochemical stainings were made on cytocentrifuge preparations using anti-LFA-1 (anti-CD 11a) and anti-ICAM-1 (anti-CD 54) monoclonal antibodies (MoAbs). Normal and sarcoid BMs displayed a high percentage of positivity with both MoAbs with no difference between study groups (LFA-1: control BM 87.8 +/- 8.8 percent; sarcoid BM 84.7 +/- 9.5 percent; ICAM-1: control BM 80.8 +/- 10 percent; sarcoid BM 88.0 +/- 4.2 percent; p = NS for all comparisons). In both groups the percentage of cells expressing LFA-1 and ICAM-1 molecules among AMs was lower than among autologous BMs (LFA-1: control AM 46.5 +/- 13.2 percent, p less than 0.001 vs control BM; sarcoid AM 64.2 +/- 15.9; p less than 0.001 vs sarcoid BM) (ICAM-1: control AM 42.7 +/- 8.5 percent, p less than 0.001 vs control BM; sarcoid AM 72.1 +/- 10.6, p less than 0.001 vs sarcoid BM). AMs from patients with PS showed a higher degree of positivity for LFA-1 and ICAM-1 than normal AMs (p less than 0.02 and p less than 0.001, respectively). The positivity for LFA-1 and ICAM-1 molecules on sarcoid AMs was not correlated with the positivity for two different BM-associated markers (ie, the CD 11b and the CD 14 molecules) and was not correlated with the percentage of T lymphocytes in BAL, selected as a marker of the intensity of the alveolitis. These results suggest that the increased ability of sarcoid AMs to induce the proliferation of T lymphocytes may be related, at least in part, to the increased expression of LFA-1 and ICAM-1 molecules on their surfaces.

Selective Enhancement of Leu Cam Expression by Interleukin 6 during Differentiation of Human Promonocytic U937 Cells

Interleukin 6 (IL-6) is a cytokine with multiple biological activities on various tissues and cells. We have investigated the effect of recombinant human IL-6 (rhIL-6) on growth and differentiation of U937. Recombinant human IL-6 induced a dose-dependent growth inhibition, apparent around day 4, of up to 50% by day 8 of culture. Concomitant with this, changes in cytochemical activities were observed, indicative of differentiation. A panel of U937 antigens was analysed after culture with rhIL-6. Expression of the majority of these membrane antigens was unaffected, with the exception of two classes. First, rhIL-6 had a profound effect on members of the leucocytic cell adhesion molecules (Leu-CAM) family. Expression of the alpha-chain of CR3 (complement receptor 3; CD11b) was enhanced in a dose-dependent fashion, with maximal expression around day 7. Parallel to this a simultaneous increase in beta-chain (CD18) expression was found. Furthermore, enhanced expression of CR3 was, accompanied by increased rosetting with sheep erythrocytes sensitized with C3bi. A much lower, but significant, enhancement was observed for the alpha-chain of the p150,95 antigen (CD11c). Expression of leucocyte function-associated antigen-1, (LFA-1), (CD11a/CD18) remained unchanged. Remarkably, however, expression of a ligand of LFA-1, intercellular adhesion molecule-1 (ICAM-1; CD54), was enhanced with similar kinetics as CR3 and p150,95. A specific anti-rhIL-6 antiserum completely inhibited the IL-6 effect. These observations provide further support for an important role of IL-6 in the regulation of myeloid cell development in man.

Leukocyte function-associated antigen-1 expression on peripheral blood mononuclear cell subsets in HIV-1 seropositive patients

In order to further investigate immune dysfunctions in HIV-1 infection, we studied the intensity of leukocyte function-associated antigen-1 (LFA-1) expression using a novel application of immunofluorescence analysis in 14 adults and 5 children seropositive for HIV-1 and in 14 healthy adults and 5 healthy children seronegative for HIV-1. While almost all lymphocytes in human peripheral blood expressed LFA-1 and while the percentage of the LFA-1 positive cells was not modified during the course of the HIV-1 infection in both adults and children, our results showed an increase of the LFA-1 expression on selected peripheral blood mononuclear cell subsets. Some LFA-1-labeled functional peripheral blood mononuclear cell subsets such as the CD16, CD14, CD3, and CD8 lymphocyte subpopulations expressed higher levels of the LFA-1 molecule during the HIV-1 infection. The LFA-1 dim cell subsets (CD4 cells) and the LFA-1 low cell subpopulation (CD19 lymphocytes) were not affected by the HIV-1 infection. Moreover, in the CD8 and CD3 cell subsets displaying a heterogeneous LFA-1 expression (dim and bright), we also observed a decrease of the LFA-1 dim LFA-1 bright cell ratio.

Differential expression of the LFA-1 molecule on the human peripheral blood mononuclear cell subpopulations

The expression of leukocyte function associated antigen 1 (LFA-1) was studied on human peripheral blood mononuclear cells (PBMNC) by a double immunofluorescence method. Monoclonal antibodies IOT18 (recognizing the beta chain, 95 kDa, CD18) and SPVL7 (recognizing the alpha chain, 180 kDa, CD11a) were used. These antibodies revealed the same heterogeneous distribution of the LFA-1 molecule among peripheral blood mononuclear cells. A bimodal profile characterized the lymphocytes, whereas the monocyte subpopulation presented a unimodal profile. NK cells, including CD16, CD8 dim, expressed the LFA-1 with a high density. B cells expressing the CD19 phenotype showed surface LFA-1 at a low level, whereas it appeared with an intermediate intensity on the T helpers/inducers (CD4 cells). Among the cytotoxic cells (CD8) and the immunoregulatory subpopulations associated with an immunosuppressive response (CD8+ CD11b+, CD8+ CD11b- and CD8+ leu8+) or with a contrasuppressive response (Vicia villosa cells), an heterogeneous LFA-1 expression was observed, discriminating the LFA-1 dim and the LFA-1 bright cells. Anti LFA-1 antibodies also allowed us to define three novel subsets among the CD8 cells: the CD8 dim LFA-1 bright, the CD8 bright LFA-1 bright, and the CD8 bright LFA-1 dim cell subpopulations. This heterogeneous distribution of LFA-1 among PBMNC may be associated with different functions of the different cell subsets analyzed in this study.

CD15 antibodies increase neutrophil adhesion to endothelium by an LFA‐1‐dependent mechanism

Anti-neutrophil antibodies (CD15) bind to a simple sugar (lactose-N-fucopentaose III, LNF III) known to be present on the chains of the adhesion molecules on neutrophils. We have demonstrated that pre-incubation of neutrophils with a CD15 antibody increases neutrophil adherence to endothelium by a neutrophil-dependent mechanism. This augmented adhesion can be inhibited by antibodies directed against the alpha and beta chain of the leukocyte function-associated antigen 1 (LFA-1) molecule. There is also some increase in surface LFA-1 expression on neutrophils after CD15 incubation, suggesting that CD15 antibodies increase neutrophil adhesion by an LFA-1-dependent mechanism. The increase in LFA-1 expression after CD15 incubation occurs in the presence of a protein synthesis inhibitor. As LFA-1 is not stored intracellularly, the increased adherence of the neutrophils, and increased LFA-1 expression on their surface, suggests the possibility that the CD15 antibodies are binding to the LNF III antigen present on the alpha and beta chains of LFA-1, producing their effect by activating the molecule, perhaps by exposing new antigen sites by a stearic effect.

In vitro modulation of leucocyte-function-associated antigen-1 expression on two leukemic cell lines.

Leukocyte-function-associated antigen-1 (LFA-1) expression on two widespread tumor cell lines: K562 (an erythroleukemia) and MOLT-4 (a T leukemia), was investigated using two monoclonal antibodies specific for the alpha chain of this surface antigen, and flow cytometry analysis. When K562 cells are in the exponential phase of growth, they display very low levels of LFA-1. By contrast, cells from the plateau phase exhibit a strong labelling, which disappears rapidly when they are allowed to resume division by changing the culture medium. Using the same experimental conditions, we failed to detect any LFA-1 expression on MOLT-4 cells. However, after stimulation of these cells by phorbol myristate acetate, we observed a significant labelling, which occurred within 2 days of treatment. The LFA-1 expression disappears progressively after removal of the phorbol ester. From these results it may be concluded that (a) LFA-1 expression can vary considerably according to the culture conditions, (b) the expression of this antigen on the surface of non-expressing variants can be induced by phorbol ester, and (c) in both cases, the change in expression can be reversed completely by replacing the culture medium or by removing phorbol myristate acetate from it.