Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet- dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.     

Endothelial leukocyte adhesion molecule 1: direct expression cloning and functional interactions.

A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokine-treated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2(+)-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.     

Tetramethylpyrazine inhibits phytohemagglutinin-induced upregulation of ICAM-1 and LFA-1 mediated leukocyte adhesion

The aim of this work was to study the effects of tetramethylpyrazine (TMP) on the interaction between lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesive molecule-1 (ICAM-1) by micropipette technique. A pair of cells, i.e., a human lymphocyte or a S KW-3 cell (human T cell leukemia), LFA-1 was expressed on which cellular surfaces, and a RBC coupled with ICAM-1, were the carriers for LFA-1 and ICAM-1. The adhesion probabilities of this pair of cells were mediated by specific interactions between ICAM-1 and LFA-1. Phytohemagglutinin (PHA) stimulation resulted in the significant increase in the adhesion probability compared to the resting lymphocyte. TMP treatment can inhibit such increase and even make the adhesion probability lower than the resting state. While the LFA-1 expression has not changed significantly with PHA stimulation or with TMP treatment, which indicated that TMP inhibiting effects was realized by a possible conformation change.     

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.     

HIV phenotype correlates with the relative amounts of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II in the virion envelope

OBJECTIVE: The biological phenotype of HIV-1 has been associated with various aspects of its infectivity, including syncytium formation and coreceptor usage. Adhesion molecules, present on both the target cell and the virus, have also been shown to play a role in the infectious process. A possible correlation between the presence of adhesion molecules in the envelope of HIV-1 with the biological phenotype of the virus is examined. DESIGN: The envelopes of 56 isolates of HIV-1 of known biological phenotype were analyzed for the presence of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II molecules. METHODS: The coreceptor usage of each isolate was determined in a GHOST cell or a U87.CD4 infectivity assay. The presence of LFA-1 and MHC class II in each virus envelope was then determined using a virus-binding enzyme-linked immunosorbent assay (ELISA). RESULTS: Viruses using the chemokine receptor CCR5 have relatively higher levels of MHC class II than LFA-1 in their envelopes compared with those using CXCR4. CONCLUSIONS: The finding that there is a differential incorporation of MHC class II and LFA-1 molecules by CXCR4- and CCR5-using viruses augments the list of properties contributing to the biological phenotype of HIV-1. This may explain, in part, how CXCR4-using viruses are able to bind to and infect a broader range of cell types than CCR5-using viruses, and why CXCR4-using viruses are associated with a more aggressive disease course.     

Control of leukocyte rolling velocity in TNF-alpha-induced inflammation by LFA-1 and Mac-1.

Previously it was shown that beta(2)-integrins are necessary for slow leukocyte rolling in inflamed venules. In this study, mice that are deficient for either one of the beta(2)-integrins, alpha(L)beta(2) (LFA-1) or alpha(M)beta(2) (Mac-1), were used to determine which of the beta(2)-integrins are responsible for slowing rolling leukocytes. The cremaster muscles of these mice were treated with tumor necrosis factor-alpha and prepared for intravital microscopy. The average rolling velocities in venules were elevated in LFA-1(-/-) mice (11.0 +/- 0.7 microm/s) and Mac-1(-/-) mice (10.1 +/- 1.1 microm/s) compared to wild-type mice (4.8 +/- 0.3 microm/s; P <.05), but were lower than in CD18(-/-) mice (28.5 +/- 2.1 microm/s). When both LFA-1 and Mac-1 were absent or blocked, rolling velocity became dependent on shear rate and approached that of CD18(-/-) mice. In addition, leukocyte adhesion efficiency was decreased in LFA-1(-/-) mice to near CD18(-/-) levels, but decreased only slightly in Mac-1(-/-) mice. Thus, both LFA-1 and Mac-1 contribute to slowing down rolling leukocytes, although LFA-1 is more important than Mac-1 in efficiently inducing firm adhesion.     

Expression of ABH and X (Lex) antigens on platelets and lymphocytes

We used a panel of reagents, polyclonal and monoclonal antibodies, and lectins to define the expression of the ABH- and Lewis-related specificities on platelets and lymphocytes. We also determined the expression of the alpha 2- and alpha 3-L-fucosyltransferases necessary for their biosynthesis. The antigens that could be detected by immunofluorescence and Western blot analysis were based on type 2 monofucosylated structures. Antibodies directed toward types 1, 3, and 4 ABH-, X- and Lewis-related antigenic determinants were always negative because the small amounts of ABH and Lewis antigens adsorbed from the serum could not be detected by these techniques. The presence of the type 2 ABH antigens on intrinsic glycoproteins was controlled by the H gene. This correlates with the presence of alpha 2-L- fucosyltransferase and the absence of alpha 3-L-fucosyltransferase on platelets. In contrast, ABH antigens were not detected by immunofluorescence on normal peripheral lymphocytes. These cells thus have only the small amounts of antigens adsorbed from the serum, these being under control of the secretor and Lewis genes. This correlates with the absence of alpha 2-L-fucosyltransferase on lymphocytes. When lymphocytes were transformed in vitro by the Epstein-Barr virus (EBV), however, they strongly expressed the X and sialylated X antigens, which are specific markers of normal granulocytes and monocytes, respectively. Treatment of EBV-transformed lymphoblastoid cell lines with 12-O-tetradecanoylphorbol-13-O-acetate significantly decreased the expression of X and sialylated X antigens along with that of surface immunoglobulins, whereas it induced a significant expression of the H antigen under control of the H gene.     

Expression of intercellular adhesion molecule 1 (ICAM-1, CD54) in colonic epithelial cells.

The expression of intercellular adhesion molecule-1 (ICAM-1, CD54) was examined in 16 surgically removed colonic tumours and two colonic carcinoma cell lines. Immunohistochemistry showed a varying percentage of ICAM-1 positive colonic carcinoma cells in 9/16 tissue specimens, while normal colonic tissue (apart from a slight reactivity of endothelial cells) was not stained. The presence of the ICAM-1 molecule on the cell surface and the expression of ICAM-1 mRNA were investigated for two colonic carcinoma cell lines. It was possible to enhance the expression of ICAM-1 considerably by incubating the cells in the presence of inflammatory cytokines in HT-29 and CaCo-2 cells. The responsiveness to either interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), or interleukin 1 beta (IL-1 beta) treatment was different in each cell line. Interestingly, ICAM-1 is shed by colonic carcinoma cells because soluble sICAM-1 was detected in the cell culture supernatants. In comparison with normal serum samples, the mean value of sICAM-1 in 63 samples of patients with colonic carcinoma and in 20 cases of active inflammatory bowel disease is raised about twofold. It remains to be clarified what part both forms of ICAM-1 play in the course of colonic cancer, ulcerative colitis, and Crohn's disease.     

Ultrastructural and functional studies on human platelets incubated with diclofenac sodium (Voltaren)

The in vitro effect of diclofenac sodium (Voltaren) on the ultrastructure of healthy donors' platelets was examined and compared with those induced by aspirin (ASA). In distinction to ASA, which causes loss of platelet pseudopodia, Voltaren induced an increase and marked elongation of these pseudopodia. The implication of this finding in the explanation of the decreased platelet aggregation caused by the drug is discussed. Voltaren increased the phagocytotic activity of the individual platelet, although the overall ability of the cells to phagocytize latex particles was not markedly increased. Platelets incubated with Voltaren showed a decrease in their total protein synthesizing capacity. Voltaren did not exert any effect on the internal ultrastructure, platelet factor 3, and calcium content of the incubated platelets.     

Investigations on the effects of heavy water (D2O) on the functional activity of human platelets

Heavy water (D2O) is able to activate or to inhibit biological systems and thus influences metabolism and functions of tissues and organs. On the basis of results obtained in preservation experiments performed on various organs, the effects of D2O on the functions of human platelets (retraction, spreading, aggregation induced by adrenaline, ADP and collagen) have been investigated. The effects of D2O, involving changes of membrane receptor conformation, binding strength of substances to their specific receptors, pH-values or enzyme activities, resulted in a significant decrease of ADP- and collagen-induced aggregation, while adrenaline-induced aggregation was significantly increased at the same D2O concentration. Retraction in the presence of D2O was significantly inhibited in a dose-related manner, while spreading was diminished, but not fully inhibited even by highest concentrations of D2O (95%).     

Expression of stanniocalcin-1 in megakaryocytes and platelets

Stanniocalcin-1 (STC) is a 56-kDa homodimeric glycoprotein hormone originally found in fish, in which it regulates calcium/phosphate homeostasis and protects against toxic hypercalcaemia. The recently characterized human STC is 80% similar to fish STC. We have earlier reported a high expression of STC in terminally differentiated human and rodent brain neurones, and found that STC contributes to the maintenance of their integrity. Here, we report that mature megakaryocytes and platelets display high STC content. K562 cells, induced to megakaryocytoid differentiation in vitro, acquired expression of STC, which was not seen in untreated K562 cells or cells induced to erythroid differentiation.     

Shedding of lymphocyte function-associated antigen-1 (LFA-1) in a human inflammatory response

Shedding of adhesion molecules has been described for members of the selectin and immunoglobulin superfamilies, but integrins are not known to be shed. Here, we describe shedding of the integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) from human leukocytes during the cutaneous inflammatory response to the blistering agent cantharidin. Expression of LFA-1 was significantly diminished on blister-infiltrated neutrophils (P < .001) and monocytes (P = .02) compared with cells in peripheral blood, but expression on lymphocytes remained unchanged. A capture enzyme-linked immunosorbent assay (ELISA) indicated that LFA-1 was shed into blister fluid as a heterodimer expressing an intact headpiece with I and I-like epitopes. However, a CD11a central region epitope, G25.2, was absent and this remained expressed as a "stub" on the cell surface of blister neutrophils. Western analysis of soluble LFA-1 revealed a truncated 110-kDa CD11a chain and a minimally truncated 86-kDa CD18 chain. However, LFA-1 was shed in a ligand-binding conformation, since it expressed KIM-127 and 24 activation epitopes and bound to solid-phase ICAM-1. Shed LFA-1 was also detected in a synovial effusion by ELISA and Western analysis. We hypothesize that LFA-1 shedding may play a role in leukocyte detachment after transendothelial migration and in regulating integrin-dependent outside-in signaling.     

Expression of intercellular adhesion molecule 1 (ICAM-1) during the development of invasion and/or metastasis of gastric carcinoma

Summary In this study, using two-color flow-cytometric analysis, we examined the expression of histocompatibility locus antigens (HLA) classes I and II, and intercellular adhesion molecule 1 (ICAM-1) in 10 cases of normal gastric mucosa, 13 cases of primary carcinoma on the stomach, 16 cases of metastatic carcinoma from malignant ascites in patients with gastric carcinoma and 14 samples of their cultured carcinoma cells. Compared with normal gastric mucosa, HLA class I were highly expressed in a considerable number of tumor cells in each experimental group. The expression of HLA class II tended to reduce in the order of normal gastric mucosa, primary gastric carcinoma and peritoneal-effusion-associated carcinoma. Altogether, 85.7% of cases of cultured tumor cells showed abrogation and loss of HLA class II. The ICAM-1 molecule was not detected on normal gastric epithelial cells. In few cases, carcinoma cells from large volumes of tumor located in the stomach showed detectable amounts of ICAM-1. On the other hand, all of the metastatic carcinoma cells from peritoneal effusions showed a high level of expression of the ICAM-1 molecule. The expression of ICAM-1 on adenocarcinoma cells was maintained and/or augmented by in vitro cultivation with tumor-infiltrating lymphocytes (TIL). Furthermore, twocolor fluorescence-activated cell sorting analysis of TIL revealed that significant correlation was observed between the expression of ICAM-1 and the degree of TIL, composed mainly of CD3⁺ T cells including CD8⁺ CD11b^–, CD8⁺CD28⁺, CD8⁺S6F1⁺ and CD4⁺Leu8⁺, and CD57⁺CD16^– and CD57⁺CD16⁺ NK cells, and HLA-DR⁺LeuM3⁺ macrophages.Abbreviations ICAM-1 intercellular adhesion molecule 1 - CTL cytotoxic T lymphocytes - TIL tumor-infiltrating lymphocytes