ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.

Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures.

In the presence of bound Mn2+, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the "open" conformation previously described only for a crystalline Mg2+ complex. The open conformation is distinguished from the "closed" form by the solvent exposure of F302, a direct T209-Mn2+ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an "active" state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.

Competition between lymphocyte function-associated antigen 1 (CD11a/CD18) and Mac-1 (CD11b/CD18) for binding to intercellular adhesion molecule-1 (CD54).

Both human integrin receptors Mac-1 (CD11b/CD18,CR3)and lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) have been demonstrated to bind human intercellular adhesion molecule-1 (ICAM-1) (CD54). Here we show that LFA-1 and Mac-1 can bind to ICAM-1 in the mouse as well. Interestingly, we observed that binding of murine LFA-1 dominates over Mac-1 for binding to ICAM-1. Using three different murine macrophage cell lines that express distinct levels of LFA-1 and Mac-1 on their cell surface, we could only detect Mac-1-dependent adhesion to ICAM-1 when little or no LFA-1 is expressed on the cell surface. When LFA-1 and Mac-1 are expressed at similar levels, the LFA-1/ICAM-1 interaction dominates over Mac-1/ICAM-1 interaction, indicating that there is a competition of LFA-1 and Mac-1 for ICAM-1 binding.

Purification and structural characterization of the CD11b/CD18 integrin alpha subunit I domain reveals a folded conformation in solution.

The alpha subunits of the leukocyte CD11/CD18 integrins contain an approximately 200 amino acid 'inserted' or I domain. The I domain of the cell-surface Mac-1 (CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM-1. The I domain from the Mac-1 alpha subunit has been expressed in Escherichia coli as a soluble GST-fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the alpha subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.

Three complement-like repeats compose the complete alpha2-macroglobulin binding site in the second ligand binding cluster of the low density lipoprotein receptor-related protein.

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of alpha2-macroglobulin (alpha2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity alpha2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for alpha2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nM) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.

Ligand specificity of purified complement receptor type three (CD11b/CD18, alpha m beta 2, Mac-1). Indirect effects of an Arg-Gly-Asp (RGD) sequence.

We have purified CR3 to homogeneity by affinity chromatography on C3bi-Sepharose and elution with EDTA. C3bi-coated erythrocytes bound to this purified CR3, and binding was dependent on the concentration of both C3bi and CR3, as well as on temperature and the presence of divalent cations. Moreover, binding could be blocked by mAb against CR3 or C3bi and could be enhanced by the addition of integrin modulating factor-1. We used the purified CR3 to test whether several putative ligands of CR3 directly bound the receptor. The interaction of purified CR3 with fibrinogen, filamentous hemagglutinin of Bordetella pertussis, lipophosphoglycan and glycoprotein 63 of Leishmania mexicana, and lipopolysaccharide from Escherichia coli was confirmed. However the interaction of CR3 with zymosan or its major component, beta-glucan, was not observed in these assays. Previous studies showed that binding of C3bi to PMN could be blocked with Arg-Gly-Asp (RGD) containing peptides and were interpreted to indicate that the RGD sequence in C3bi interacts directly with CR3. We found, however, that RGD containing peptides were unable to block the interaction of C3bi with purified CR3, yet retained the ability to block binding of C3bi to cells. We conclude that RGD-peptides do not directly bind CR3, but instead indirectly effect CR3 function. Inasmuch as the effect of RGD-peptides could be mimicked with antibodies against leukocyte response integrin, we suggest that RGD-peptides may bind to leukocyte response integrin on polymorphonuclear leukocytes and influence CR3 activity via this receptor.

Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo.

At day 10 in mouse gestation, the intraembryonic aorta-gonads-mesonephros (AGM) region generates the first definitive hematopoietic stem cells (HSCs) of the adult blood system. By 11 days postcoitum, the liver contains such HSCs. While HSCs of the adult bone marrow and late-stage fetal liver have been extensively characterized for cell surface markers, there has been no phenotypic description of the first HSCs during embryo development. We report here the temporal cell surface phenotype of HSCs from the AGM region and early fetal liver and show that all HSCs reside in the c-kit+ population. c-kit+ HSCs from AGM and liver are mainly CD34+ and in the AGM are in both Mac-1+ and Mac-1 fractions. These results demonstrate that during mouse ontogeny the first definitive HSCs are similar in cell surface phenotype to the HSCs of adult bone marrow but that spatial localization and developmental time are critical factors in the phenotypic assessment of this functional cell population.

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis.

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. with Cryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyte- and granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogeneously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.

Essential role of interleukin-12 (IL-12) and IL-18 for gamma interferon production induced by listeriolysin O in mouse spleen cells.

The mechanism of gamma interferon (IFN-gamma) production induced by listeriolysin O (LLO), a cytolytic virulence factor of Listeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant of L. monocytogenes was capable of inducing a high level of IFN-gamma when its cytolytic activity was blocked by cholesterol treatment. The IFN-gamma-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b(+) cells resulted in a profound decrease in IFN-gamma production in response to LLO stimulation. Negative selection also suggested the contribution of DX5(+) cells in IFN-gamma production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b(+) fraction of spleen cells was unchanged. There was no change in the expression of the IFN-gamma-inducing cytokine genes in the CD11b(-) fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-gamma production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-gamma after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor of L. monocytogenes, is capable of inducing IFN-gamma from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host.

The role of interferon-gamma in murine pneumococcal pneumonia.

To determine the role of interferon (IFN)-gamma in pneumonia, IFN-gamma receptor-deficient (IFN-gamma R(-/-)) and 129/Sv (wild-type [wt]) mice were inoculated intranasally with Streptococcus pneumoniae. Although mortality did not differ between the groups 48 h after inoculation, IFN-gamma R(-/-) mice had significantly fewer pneumococci in their lungs than the wt mice. Similarly, IFN-gamma(-/-) mice had fewer colony-forming units in lungs than wt mice. The relatively increased resistance of IFN-gamma R(-/-) mice was not related to favorable effects on defense mechanisms known to contribute to antibacterial immunity-that is, the neutrophilic influx was reduced and the cytokine and nitric oxide levels were similar or lower in IFN-gamma R(-/-) mice. In contrast, mice treated with anti-IFN-gamma did not demonstrate a consistently altered bacterial outgrowth, compared with mice treated with a control antibody. These data suggest that endogenous IFN-gamma, despite its protective role in defense against intracellular pathogens, does not serve a protective role during pneumococcal pneumonia.

Characterization of C3 receptors on cultured rat glomerular endothelial cells.

In this study we characterized C3 receptors on cultured rat glomerular endothelial cells (GEnC), using immunochemical and molecular techniques. GEnC membrane proteins were immunoprecipitated with a polyclonal antibody directed towards mouse complement receptor 2 (CR2). This anti-MCR2 immunoprecipitated GEnC proteins of 120 and 150 kDa. By immunohistochemistry, anti-MCR2 stained GEnC in rat glomeruli in vivo. Given the presence of CR2-like proteins on GEnC, subsequent studies were done to determine whether GEnC had C3-binding proteins. GEnC proteins of 80, 200, and 300 kDa specifically bound to columns of rat C3d-Sepharose and C3b-Sepharose, illustrating that these proteins were binding to the C3d portion of C3. The 80, 200, and 300 kDa C3d-binding proteins were distinct from the 120 and 150 kDa anti-MCR2 reactive proteins, as shown by immunoabsorption studies. Next, a specific cDNA probe for rat CR2 was generated by RT-PCR. Oligonucleotides were chosen from highly conserved regions in mouse and human CR2 spanning 224 bases, with the rationale that these would also be conserved in the rat. A 224 bp PCR product was generated from both rat GEnC and rat kidney cDNA, illustrating the presence of CR2 mRNA in these tissues. By Northern analysis, the CR2 PCR product hybridized to mRNA of 2 and 5 kb from GEnC. The 5 kb transcript was also identified in rat kidney mRNA. Therefore, proteins immunologically related to mouse CR2 are present in GEnC in vitro and in vivo. C3d-binding proteins of 80, 200, and 300 kDa are also present on rat GEnC, yet these appear to be immunologically distinct from the proteins identified by anti-MCR2. Whether the GEnC CR2 mRNA transcripts of 2 and 5 kb are translated into the 80 and 200 kDa C3d-binding proteins or the 120 and 150 kDa mouse CR2-like proteins remains to be defined.

AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-gamma receptor-deficient mice.

Autoimmune collagen-induced arthritis (CIA) in IFN-gammaR-deficient DBA/1 mice was shown to be reduced in severity by treatment with the bicyclam derivative AMD3100, a specific antagonist of the interaction between the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4. The beneficial effect of the CXCR4 antagonist was demonstrable when treatment was initiated between the time of immunization and appearance of the first symptoms. Treatment also reduced the delayed-type hypersensitivity response to the autoantigen, collagen type II. These observations are indicative of an action on a late event in the pathogenesis, such as chemokine-mediated attraction of leukocytes toward joint tissues. The notion of SDF-1 involvement was further supported by the observation that exogenous SDF-1 injected in periarthritic tissue elicited an inflammatory response that could be inhibited by AMD3100. The majority of leukocytes harvested from inflamed joints of mice with CIA were found to be Mac-1(+) and CXCR4(+), and AMD3100 was demonstrated to interfere specifically with chemotaxis and Ca(2+) mobilization induced in vitro by SDF-1 on Mac-1(+)/CXCR4(+) splenocytes. We conclude that SDF-1 plays a central role in the pathogenesis of murine CIA, by attracting Mac-1(+)/CXCR4(+) cells to the inflamed joints.

Lymphocytes utilize CD11b/CD18 for adhesion to Candida albicans.

Large granular lymphocytes require adherence to hyphae of Candida albicans to inhibit growth of this fungus. This study was undertaken to identify the lymphocyte surface structures that mediate this adhesion. Monoclonal antibodies specific for epitopes of the alpha subunit (CD11b) and the beta 2 subunit (CD18) of Mac-1 eliminated lymphocyte adhesion to C. albicans hyphae. Significant inhibition of lymphocyte adhesion to C. albicans was also achieved with known protein ligands of Mac-1. These proteins included the extracellular matrix proteins vitronectin, laminin, and fibrinogen as well as two engineered peptides containing RGD (arginine-glycine-aspartic acid) sequences. Carbohydrates including N-acetyl-D-glucosamine which have been demonstrated to inhibit Mac-l-mediated adhesion to whole yeast and yeast zymosan also blocked lymphocyte adhesion to hyphae. These results identify Mac-1 (CD11b/CD18) as the surface structure that mediates lymphocyte adhesion to C. albicans. A model is proposed for lymphocyte Mac-1 activation by microbial ligands.

Human polymorphonuclear leukocytes adhere to complement factor H through an interaction that involves alphaMbeta2 (CD11b/CD18).

The work presented here demonstrates that human complement factor H is an adhesion ligand for human neutrophils but not for eosinophils. The adherence of polymorphonuclear leukocytes (PMNs) to plastic wells coated with factor H depended on divalent metal ions and was augmented by C5a and TNF-alpha. PMN adhesion to factor H in the presence or absence of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity purification using factor H Sepharose followed by immunoprecipitation using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as a factor H binding receptor. The presence of surface bound factor H enhanced neutrophil activation resulting in a two- to fivefold increase in the generation of hydrogen peroxide by PMNs stimulated by C5a or TNF-alpha. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells adhered to immobilized heparin or chondroitin A. In addition, augmented adhesion of PMNs was measured when factor H, but not HSA or C9, was absorbed to wells that were first coated with heparin or chondroitin A. The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs to CD11b and CD18. These studies demonstrate that factor H is an adhesion molecule for human neutrophils and suggest that the interaction of factor H with glycosaminoglycans may facilitate the tethering of this protein in tissues allowing factor H to serve as a neutrophil adhesion ligand in vivo.