Leukocyte receptor expression in chronic periodontitis.

BACKGROUND AND OBJECTIVE: Microbial recognition in the periodontium through specific leukocyte receptors gives rise to the response which in susceptible individuals can lead to periodontal diseases. The aim of this study was to explore the expression of leukocyte receptors in the gingival tissues of chronic periodontitis patients and to analyse differences between diseased and control sites (sites with probing pocket depth <4 mm). MATERIAL AND METHODS: Thirty-seven chronic periodontitis patients were included in the study. Gingival biopsies were harvested from diseased and control sites and processed by flow cytometry for the determination of the expression of 16 leukocyte receptors (CD4, CD8, CD11b, CD14, CD16, CD19, CD25, CD28, CD49d, CD49e, CD62, CD71, CD80, CCR7, Ly6G and HLA-DR). RESULTS: Expression of all studied receptors was higher in test compared with control sites (p < 0.005). Sampled sites with less bleeding on probing exhibited higher expression of CD16 and CD14 receptors (p = 0.020 and 0.011, respectively). CONCLUSIONS: This study points towards considerable differences in the expression of leukocyte receptors between diseased and control sites in the same periodontal patients.

Human neutrophil adherence to laminin in vitro. Evidence for a distinct neutrophil integrin receptor for laminin.

We used mAbs against polymorphonuclear leukocyte (PMN) surface proteins to investigate the mechanisms by which stimulated human neutrophils (PMNs) adhere in vitro to laminin, the major glycoprotein of mammalian basement membrane. mAb IB4, which is directed against the common beta 2 chain of the CD11/CD18, only partially inhibited the adherence of PMA-stimulated PMNs to both laminin and to subendothelial matrices. In contrast, IB4 completely inhibited PMA-stimulated PMN adherence to gelatin, fibronectin, collagen IV, and endothelial cell monolayers. PMA-stimulated PMNs from a patient with severe congenital CD11/CD18 deficiency also adhered to laminin, but not to gelatin or endothelial cell monolayers. Therefore, PMA-stimulated PMNs adhere to laminin by both CD11/CD18-dependent and CD11/CD18-independent mechanisms. Expression of CD11/CD18-independent adherence to laminin was agonist dependent, occurring after stimulation with the calcium ionophore A23187 and recombinant TNF-alpha, but not with the chemotactic factors FMLP, platelet activating factor, or recombinant human C5a. Expression of CD11/CD18-independent adherence was also divalent cation dependent, occurring in the presence of Mg2+ but not Ca2+ as the sole added divalent cation. The mAbs AIIB2 and 13, which are directed against the beta 1 subunit of the VLA integrins, significantly inhibited the CD11/CD18-independent adherence of normal PMNs to laminin, and completely abolished the adherence of CD11/CD18-deficient PMNs to laminin. Both anti-beta 1 mAbs bound to PMNs, as demonstrated by flow cytometry, and immunoprecipitated a membrane molecule of Mr 130,000 daltons from 125I-labeled, detergent-solubilized PMNs. These data suggest that human PMNs possess beta 1 and beta 2 classes of integrins, and that both mediate PMN adherence.

Leukocyte response integrin and integrin-associated protein act as a signal transduction unit in generation of a phagocyte respiratory burst.

The leukocyte response integrin (LRI) is a phagocyte integrin which recognizes the basement membrane protein entactin and the synthetic peptide Lys-Gly-Ala-Gly-Asp-Val (KGAGDV). The function of LRI is intimately associated with that of a distinct membrane protein, integrin-associated protein (IAP), as antibodies which recognizes IAP can inhibit all known functions of LRI. When adherent to surface, the LRI ligands entactin and KGAGDV activate the respiratory burst in polymorphonuclear leukocytes (PMN) and monocytes, as do monoclonal antibodies (mAb) directed at either LRI or IAP. When added in solution, peptides and antibodies specific for LRI, and some, but not all, anti-IAP antibodies, can inhibit the respiratory burst activated by any of these surface-adherent ligands. Only monoclonal anti-IAP antibodies which can inhibit LRI function when added in solution are competent to activate the respiratory burst when adherent to a surface. KGAGDV peptide and anti-LRI added in solution can inhibit anti-IAP-stimulated respiratory burst. The LRI-IAP-initiated respiratory burst is independent of CD18, as judged by: (a) blockade of inhibition by anti-CD18 mAb with the protein kinase A inhibitor HA1004; (b) enhanced sensitivity of CD18-dependent respiratory burst compared with LRI/IAP-dependent respiratory burst to the tyrosine kinase inhibitors genestein and herbimicin; and (c) generation of a respiratory burst in response to KGAGDV, anti-LRI, and anti-IAP coated surfaces in PMN from a patient with LAD. Despite its apparent CD18 independence, LRI/IAP-initiated respiratory burst requires a solid phase ligand and is sensitive to cytochalasin B. These data suggest a model in which LRI and IAP act together as a single signal transduction unit to activate the phagocyte respiratory burst, in a manner that requires CD18-independent cell adhesion.

Crosslinking of the T cell-specific accessory molecules CD7 and CD28 modulates T cell adhesion.

Regulated adhesion enables T cells to migrate through tissue and transiently interact with an endless succession of cells. Monoclonal antibody (mAb) engagement of the CD3/T cell receptor (TCR) complex results in a rapid and transient augmentation of the adhesion function of LFA-1 and VLA integrin molecules on human T cells. We show in this study that mAb crosslinking of the T cell-specific accessory molecules CD7 and CD28, or treatment with the Ca2+ ionophore A23187, results in the rapid induction of integrin-mediated adhesion to three distinct ligands: the extracellular matrix protein fibronectin, and the cell surface molecules ICAM-1 and VCAM-1. Like CD3 crosslinking, increased adhesion via CD7 and CD28 crosslinking appears to involve both protein kinase C (PKC) and cAMP-dependent protein kinases. In contrast, A23187 induction of adhesion is unaffected by PKC inhibitors. CD7 is preferentially expressed on naive T cells and is unique in being a potent inducer of naive T cell adhesion. Enhanced expression/function of adhesion-inducing molecules thus overcomes relative deficits in adhesion receptor expression.

Inducible cell adhesion molecule 110 (INCAM-110) is an endothelial receptor for lymphocytes. A CD11/CD18-independent adhesion mechanism.

Inducible cell adhesion molecule 110 (INCAM-110) is a 110-kD glycoprotein expressed on cytokine-activated human vascular endothelial cells. mAb blocking studies indicate that INCAM-110 and intercellular adhesion molecule 1 (ICAM-1) independently support the adhesion of lymphocytes to activated human umbilical vein endothelial cell monolayers. Anti-CD11a/CD18 antibodies with anti-INCAM-110 mAb E1/6 produce greater inhibition of lymphocyte adhesion than either reagent alone, suggesting that INCAM-110 and LFA-1 are not an obligate receptor-ligand pair. Blood monocytes, but not polymorphonuclear leukocytes, also appear to bind endothelial INCAM-110. Endothelial expression of INCAM-110 is upregulated at sites of inflammation, suggesting a role in the recruitment of mononuclear leukocytes.

Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.

The cytokine NAP-1/IL-8 is produced by a variety of different cells in response to inflammatory stimuli and elicits several biological responses from PMN. Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged. Increased CD11b/CD18 and CD11c/CD18 appears to correspond with the release of specific granules by NAP-1/IL-8. NAP-1/IL-8 was also a potent stimulator of several of the binding activities of CD11b/CD18. Ligation of EC3bi by CD11b/CD18 was rapidly enhanced by NAP-1/IL-8, but phagocytosis of the ligated particles was not induced by the agonist. In addition, enhanced binding of EC3bi was observed in the absence of an increase in receptor expression as shown with PMN cytoplasts. NAP-1/IL-8 promoted additional adhesive interactions between CD11b/CD18 and the biosynthetic precursor of LPS, lipid IVa, fibrinogen, and endothelial cells, suggesting that NAP-1/IL-8 may promote leukocyte adhesion in vivo that could lead to recruitment of PMN to sites of tissue inflammation.

Differential roles of Mac-1+ cells, and CD4+ and CD8+ T lymphocytes in primary nonfunction and classic rejection of islet allografts.

The high rate of persistent hyperglycemia, termed primary nonfunction, after islet allotransplantation in C57BL/6 mice recipients of B10.BR strain islets, as compared with B10.BR recipients of C57BL/6 islets, led to a series of experiments to determine whether islet allograft primary nonfunction was attributable to technical aspects of the transplant procedure or whether it was a consequence of alloimmunity. Primary nonfunction was prevented by systemic pharmacologic immunosuppression of the host with cyclosporine. Selective immunodepletion of host CD4+ and CD8+ T lymphocytes significantly extended the time of classic rejection but did not significantly affect the rate of primary nonfunction. However, modulation of macrophages by administration to the host of silica completely abolished primary nonfunction. These observations, in conjunction with the immunohistological findings of intense macrophage infiltration in islet allografts from recipients exhibiting persistent post-transplant hyperglycemia, support the hypothesis that primary nonfunction results from a cell-mediated host-immune response of rapid onset that is dependent on macrophages or macrophage byproducts as the main effectors.

Constitutive and stimulus-induced phosphorylation of CD11/CD18 leukocyte adhesion molecules.

The leukocyte CD11/CD18 adhesion molecules (beta 2 integrins) are a family of three heterodimeric glycoproteins each with a distinct alpha subunit (CD11a, b, or c) and a common beta subunit (CD18). CD11/CD18 mediate crucial leukocyte adhesion functions such as chemotaxis, phagocytosis, adhesion to endothelium, aggregation, and cell-mediated cytotoxicity. The enhanced cell adhesion observed upon activation of leukocytes is associated with increased surface membrane expression of CD11/CD18, as well as a qualitative upregulation of CD11/CD18 functions. To elucidate the nature of the qualitative modifications that occur, we examined the phosphorylation status of these molecules in resting human leukocytes and upon activation with PMA or with the chemotactic peptide F-met-leu-phe (FMLP). In unstimulated cells, all three CD11 subunits were found to be constitutively phosphorylated. In contrast, phosphorylation of the common CD18 subunit was minimal. PMA induced rapid and sustained phosphorylation of CD18 that occurred at high stoichiometry, but had only minimal effects on phosphorylation of the associated CD11 subunits. FMLP also induced rapid phosphorylation of CD18, but the effect was of short duration. FMLP-induced phosphorylation of CD18 was not related to its Ca++-mobilizing effect, as CD18 phosphorylation was not observed upon treatment of leukocytes with the Ca++ ionophore, ionomycin. Phosphoamino acid analysis of CD11/CD18 in PMA- or FMLP-treated monocytes revealed a predominance of phosphoserine residues in all CD11/CD18 subunits. A small component of phosphothreonine was present in CD11c and CD18 and a minor component of phosphotyrosine was also detected in CD18 upon leukocyte activation may regulate the adhesion functions mediated by the CD11/CD18 family of molecules.

CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.

Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

Prevention of activated neutrophil adhesion to endothelium by soluble adhesion protein GMP140.

Neutrophils and monocytes, but not lymphocytes, adhered strongly to plastic surfaces coated with GMP140, a protein of endothelial cells and platelets. This adhesion of neutrophils was mediated by GMP140 and not by the CD18 integrin complex. By contrast, GMP140 in solution inhibited the CD18-dependent adhesion of tumor necrosis factor-alpha-activated neutrophils to plastic surfaces and resting endothelium, but not of resting neutrophils to tumor necrosis factor-alpha-activated endothelium. Thus, the binding of a soluble form of an adhesion protein selectively inhibited another set of adhesive events. Soluble GMP140 may be important in maintaining the nonadhesiveness of neutrophils in the circulation and may serve to limit inflammatory reactions.

Correction of CD18-deficient lymphocytes by retrovirus-mediated gene transfer.

Leukocyte adhesion deficiency (LAD) is an inherited disorder of leukocyte function caused by derangements in CD18 expression. The genetic and functional abnormalities in a lymphocyte cell line from a patient with LAD have been corrected by retrovirus-mediated transduction of a functional CD18 gene. Lymphocytes from patients with LAD were exposed to CD18-expressing retrovirus and enriched for cells that express CD11a and CD18 (LFA-1) on the cell surface. Molecular and functional analyses of these cells revealed (i) one copy of proviral sequence per cell, (ii) viral-directed CD18 RNA that exceeded normal endogenous levels, (iii) normal quantities of CD11a and CD18 protein on the cell surface, and (iv) reconstitution of LFA-1-dependent adhesive function.

The acute inflammatory reaction.

A major theme in current developments in this field has been the realization that the target tissue, in particular the endothelium, has an active role in initiating and participating in inflammatory reactions and is not a passive target, as was previously thought. Attempts to delineate the mechanisms involved have benefited immeasurably from the application of the modern techniques of molecular and cellular biology, and will continue to do so. It is axiomatic that a fuller understanding of these mechanisms will result in the development of new strategies designed to block the acute inflammatory response more specifically and effectively.

The pancreatic islet endothelial cell: emerging roles in islet function and disease.

The pancreatic islets are one of the most vascularized organs of the body. This likely reflects the requirements of the organ for a rich supply of nutrients and oxygen to the tissue, as well as the need for rapid disposal of metabolites and secreted hormones. The islet endothelium is richly fenestrated to facilitate trans-endothelial transport of secreted hormones, has a unique expression of surface markers, and produces a number of vasoactive substances and growth factors. The islet endothelial cells play a critical role in the early phase of type 1 diabetes mellitus by increasing the expression of surface leucocyte-homing receptors, thereby enabling immune cells to enter the endocrine tissue and cause beta-cell destruction. Following transplantation, pancreatic islets lack a functional capillary system and need to be properly revascularized. Insufficient revascularization may severely affect the transport properties of the islet endothelial system, resulting in a dysfunctional islet graft.

Polyunsaturated fatty acids interfere with formation of the immunological synapse.

Polyunsaturated fatty acids (PUFAs) exert inhibitory effects on T cell-mediated immune responses. Activation of T cells in vivo depends on formation of an immunological synapse (IS) at the T cell/antigen-presenting cell (APC) interface. Here, we analyzed effects of PUFA treatment on the formation of the IS and APC-induced human T cell activation. In T cells treated with the PUFA eicosapentaenoic (EPA; 20:5,n-3) and arachidonic acid (20:4,n-6), stimulated by superantigen-presenting cells or APCs, relocalization to the IS of distinct molecules [F-actin, talin, leukocyte functional antigen-1alpha, clusters of differentiation (CD)3epsilon] was inhibited markedly compared with cells treated with saturated fatty acid, whereas relocalization of protein kinase Ctheta to the IS remained unaffected. CD3-induced, sustained phosphorylation of nucleotide exchange factor Vav, which controls cytoskeletal rearrangements underlying IS formation, was significantly reduced in EPA-treated Jurkat and peripheral blood T cells. In addition, T cell raft disruption by methyl-beta-cyclodextrin treatment and experiments with a chimeric linker for activation of T cell proteins, which is resistant to PUFA effects on lipid rafts, revealed modifications of lipid rafts as a crucial factor for PUFA-mediated inhibition of APC-stimulated cytoskeletal rearrangements. Furthermore, the efficiency of T cell/APC conjugate formation was significantly reduced with EPA-treated T cells, as was stimulation of CD69 expression, which is not altered following antibody-mediated T cell activation. In conclusion, PUFA treatment of T cells qualitatively and quantitatively alters IS formation, thereby extending T cell signaling defects to pathways that are not intrinsically altered in PUFA-treated T cells when stimulated by antibodies.

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18).

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzyme-histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.

Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.

Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs.

The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 microgram/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5-45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 micrograms/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10(-5) M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant. These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.

Fc gamma RII-mediated superoxide production by phagocytes is augmented by GM-CSF without a change in Fc gamma RII expression.

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.

Monoclonal antibodies against porcine LFA-1: species cross-reactivity and functional effects of beta-subunit-specific antibodies.

As a first step in developing a porcine model system to study the effect of LFA-1-specific monoclonal antibodies on allograft rejection, we have identified anti-human LFA-1 monoclonal antibodies that cross-react with porcine leukocytes and have used one of these antibodies against the LFA-1 beta-subunit to purify porcine LFA-1. Immunization of Balb/c mice with the purified antigen yielded eight monoclonal antibodies directed against the beta-subunit of porcine LFA-1. These antibodies immunoprecipitated pig proteins corresponding to the human alpha-subunits of LFA-1 (CD11a), Mac-1 (CD11b) and LeuM5 (CD11c), and their common beta-subunit (CD18). Under both reducing and non-reducing conditions, the porcine beta-subunit showed a different electrophoretic mobility from that of the human beta-subunit. In addition, results of peptide mapping studies revealed structural differences between the beta-subunits of human and porcine LFA-1. The anti-LFA-1 beta-chain antibodies were found to cross-react with antigens expressed on rat, mouse, hamster, rabbit, dog, cow and human leukocytes, indicating that the beta-subunit contains highly conserved epitopes. Competitive binding studies showed that these antibodies and three anti-human LFA-1 antibodies defined at least four distinct epitopes on the porcine beta-subunit, three of which were also found on human beta-subunits. Whereas the cross-reactive anti-human beta-subunit antibody H52 profoundly inhibited mitogenic and allogenic stimulation of porcine T lymphocytes as well as cytotoxic T cell and natural killer (NK) cell function, the anti-porcine LFA-1 monoclonal antibodies had very limited effect on porcine lymphocyte function. Our results demonstrate that porcine leukocytes express the LFA-1 molecule and that this antigen is involved in T cell and NK cell functions, as has previously been shown in human and murine systems. We conclude that the pig may serve as a useful model in which to test the effect of anti-LFA-1 antibodies on allograft rejection.

Production and characterization of monoclonal anti-CD18 anti-idiotype antibodies.

Three leukocyte adhesion receptors have been described which mediate intercellular binding of leukocytes: LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150/95 (CD11c/CD18). We have previously reported the production of several monoclonal antibodies against the common subunit of these receptors (CD18). We have describe the production of monoclonal anti-idiotype antibodies against one of the anti-CD18 antibodies (H52) which has been shown to inhibit potently the function of leukocyte adhesion receptors. Three IgG1 and two IgM anti-idiotype antibodies were derived which recognized private idiotopes on the H52 molecule. Two of these antibodies blocked the binding of H52 to purified LFA-1 and to cell surface expressed antigen. One of the antibodies (AIM.6) was shown to be an internal image-type (Ab2 beta) antibody based on inhibition of its binding to H52 by purified LFA-1 and by its ability to induce Ab3 which recognize LFA-1 when used as immunogen. The AIM.6 Ab2 beta antibody was tested for recognition of leukocyte adhesion ligands in LFA-1-mediated leukocyte adhesion and activation assays. The AIM.6 antibody did not block intercellular adhesion of leukocytes or mitogen stimulation of T cells, functions which were completely inhibited by low concns of H52. AIM.6 Ab2 beta antibody bound to H52 very well at 0 degrees C but bound very poorly or not at all at 37 degrees C. Binding studies on a panel of anti-CD18 monoclonal antibodies showed that the idiotope defined by AIM.6 was unique to H52 and an antibody recognizing the same epitope on CD18 (H5B9). This result showed that inhibitory anti-CD18 monoclonal antibodies utilize at least two distinct paratopes in binding to CD18. The above results are in contrast to those obtained in other systems in which Ab2 beta antibodies against receptor-specific Ab1 antibodies recognize receptor ligands and are discussed in the context of ligand recognition by leukocyte adhesion receptors.