A role for Ia antigens in thymocyte binding by macrophages

To investigate the membrane structures involved in cellular interactions between thymocytes and macrophages, the relative ability of different murine macrophage populations to spontaneously bind thymocytes was compared. Macrophages derived from the spleen or thymus bound three to four times the number of thymocytes than macrophages from peripheral blood, peritoneum, or bone marrow. This reflects differences both in the number of macrophages binding thymocytes and in the number of thymocytes bound per macrophage. The extent of binding seems to positively correlate with the number of Ia-positive macrophages contained in these populations, as based on previously published values. This was confirmed by showing that elimination of splenic Ia-positive macrophages with anti-Ia and complement treatment dramatically reduced thymocyte binding. In addition, mouse peritoneal washout macrophages incubated for several days with supernatant fluid from concanavalin A-stimulated spleen cells, which induce Ia-antigen expression, exhibited a marked increase in the number of macrophages that bound thymocytes and the number of thymocytes bound per macrophage. To determine if Ia antigens were directly involved in binding, spleen, thymus, or Ia-induced peritoneal macrophages were treated with a monoclonal anti-Ia antibody prior to the addition of thymocytes. Treatment with anti-Ia reduced binding by around 50%, whereas treatment with anti-H-2D antibody had no effect. Monoclonal anti-I-A and anti-I-E antibody treatments of macrophages both inhibited thymocyte binding to similar extents, and treatment of macrophages with both reagents together reduced thymocyte binding by 80%. These results indicate that thymocyte binding is in part dependent on macrophage Ia expression.     

Novel cell surface antigens expressed on mouse alveolar macrophages.

Two new cell surface antigens expressed on mouse alveolar macrophages were defined by rat monoclonal antibodies. One marker, AVM-1, was detected on mouse alveolar macrophages, but it was undetectable on resident peritoneal cells, thioglycollate medium-induced peritoneal cells, and splenic macrophages. Splenic lymphocytes, thymocytes and bone marrow cells were also AVM-1 negative. Anti-AVM-1 monoclonal antibody immunoprecipitated a single polypeptide with a molecular weight of 200,000. Of particular interest was the finding that the anti-AVM-1 antibody could inhibit the formation of EA and EAC rosette on macrophage line cells. A second antigen (AVM-2) was also present on alveolar macrophages, and its molecular weight was 38,000.     

A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on the differentiation and functional capacity of macrophages cultured in vitro.

The effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) on the differentiation and functional capacity of macrophages cultured in vitro from various lymphoid tissues was investigated. In cultures of peritoneal cells, the number of macrophages did not change throughout incubation periods of from 1 hr to 3 days, and the addition of CPS-K had no affect. It appears therefore that CPS-K does not exhibit cytotoxic effects on macrophages. In cultures of spleen cells, only a small number of macrophages appeared within 1 hr, but the number of macrophages increased during further incubation. The addition of CPS-K to cultures of spleen cells at the start of incubation suppressed markedly the increase in the numbers of macrophage. This finding indicates that CPS-K blocks the process of the generation of macrophages, probably from their precursor cells in cultures of spleen cells. Only a small number of macrophages appeared in cultures of thymocytes or lymph node cells either with or without CPS-K. The phagocytic capacity of either peritoneal macrophages or macrophages generated in cultures of spleen cells was activated during incubation in vitro. Macrophages cultured in the presence of CPS-K for 24 hr or longer appeared to have an enhanced phagocytic activity, although the enhancement of their phagocytic activity by the addition of CPS-K was less marked in cultures of spleen cells than in those of peritoneal macrophages. Morphologically, macrophages in both cultures of peritoneal cells and spleen cells incubated in the presence of CPS-K for 4 days possessed much longer cytoplasmic processes than those incubated in the absence of CPS-K. From the present study, it appears that CPS-K exhibits dual effects on macrophage precursor cells and macrophages, a blocking effect on the differentiation from the former to the latter and an enhancing effect on the functional capacity of the latter.     

Macrophage activation of allogeneic lymphocyte proliferation in the guinea pig mixed leukocyte culture.

The role of the macrophage in the guinea pig mixed leukocyte culture was investigated. Macrophages obtained from oil-induced peritoneal exudates, peritoneal wash-out cells, spleen, and alveolar washings were found to be effective stimulators of allogeneic lymph node and splenic lymphocyte DNA synthesis. The stimulatory properties of macrophages proved radioresistant but viability dependent. Unfractionated lymph node cells or adherence column purified lymph node lymphocytes and thymocytes were only minimally active as stimulators, even in the presence of macrophages syngeneic to the responder lymphocytes. Allogeneic fibroblasts, polymorphonuclear leukocytes, L2C leukemia cells, and xenogeneic (murine) macrophages failed to simulate. These data provide evidence that the macrophage is the predominant stimulator of the mixed leukocyte culture in the guinea pig.     

Leishmania enriettii: Immune induction of macrophage activation in an experimental model of immunoprophylaxis in the mouse

This study describes some of the parameters of the cellular immune response elicited in mice by inoculation of the nonpathogenic protozoan parasite, Leishmania enriettii. Incubation in vitro of leishmania-infected mouse peritoneal macrophages with spleen cells from syngeneic leishmania-immune animals resulted in activation of the phagocytes, leading to intracellular parasite destruction. Activation required interaction of sensitized lymphocytes with parasite antigen released or displayed by infected macrophages. The effect was dependent both on the dose of parasites used for in vivo priming and on the number of spleen cells cocultivated with parasitized macrophages. The activating capacity of lymphocytes was abrogated by anti-Thy-1 antiserum treatment and was retained in the effluent cells after nylon-wool separation. Activation was followed by lysis of part of the macrophage monolayer. Destruction of the phagocytes did not appear to result from the activation process per se and may represent a cytotoxic activity of sensitized lymphocytes for macrophages bearing parasite antigen on their surface.     

Biosynthesis of C4 by mouse peritoneal macrophages. II. Comparison of C4 synthesis by resident and elicited cell populations

Five elicited macrophage populations synthesized one-third to one-tenth as much hemolytically active C4 when compared with resident peritoneal macrophages. This decrease in functional C4 activity was not caused by inhibitors or protease activity in the elicited macrophage supernatants. Analysis of C4 antigen indicated a similar reduction in the elicited cells compared with the resident macrophages. A defect in precursor processing or secretion was deemed unlikely because the intracellular C4 precursor was appropriately reduced in the elicited cells. We postulate that mouse resident peritoneal macrophages are a pluripotent cell population with broad capabilities in regard to the initiation of the inflammatory response. In contrast, elicited or activated macrophages may be more specialized cells, with one manifestation of this being the "down" regulation of C4 synthesis.     

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.     

Mouse macrophage hybridomas secreting a cytotoxic factor and interleukin 1

Stable mouse macrophage hybridomas were produced by somatic cell fusion between proteose peptone-elicited peritoneal macrophages and NS-1 myeloma cells. Three cloned hybrid cell lines, designated as N/P-5-3, -6-2, and -7-1, exhibited typical macrophage-like morphology. Moreover, their macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mac-1 antigens and Fc-receptors on the cell surface, and the demonstration of phagocytic and antigen-presenting activities. Furthermore, these cell lines, stimulated with LPS, secreted considerable amounts of a cytotoxic factor and interleukin 1. Cultured cells of various tumor cell lines were sensitive to the cytotoxic factor, but normal thymocytes, spleen cells, and liver cells were not killed by the factor.     

Effects of differentiation in vivo and of lymphokine treatment in vitro on the mobility of C3 receptors of human and mouse mononuclear phagocytes

The C3 receptors of human peripheral blood monocytes are able to move laterally within the plasma membranes of the cells and remain mobile even when the cells develop into "macrophages" in vitro. In contrast, the C3 receptors of mouse peritoneal macrophages are immobile. To determine whether these differences are species differences or differences between cells of different stages of differentiation, we assessed the mobility of C3 receptors of mouse peripheral blood monocytes and of human pulmonary alveolar and peritoneal macrophages. The C3 receptors of mouse monocytes were mobile, whereas the C3 receptors of human tissue macrophages were immobile. The C3 receptors of macrophages mediate avid particle binding but do not normally promote ingestion. We have described a unique lymphokine that activates mouse peritoneal macrophage C3 receptors for phagocytosis by freeing them from their plasma membrane anchors. In the present experiments, we found that the lymphokine also freed the C3 receptors of human macrophages and activated them for phagocytosis. We conclude that the immobilization of C3 receptors appears to be a marker for the differentiation of human and mouse mononuclear phagocytes, that the differentiation of mononuclear phagocytes is influenced by the milieu in which the cells develop, that in vitro-differentiated macrophages may not accurately represent tissue macrophages, and that a lymphokine activates the C3 receptors of both human and mouse macrophages for phagocytosis by allowing the receptors lateral mobility within the cell plasma membrane.     

Inducible expression of murine IP-10 mRNA varies with the state of macrophage inflammatory activity

We have examined the expression of inducible inflammatory genes in murine macrophages from different tissues and at different stages of inflammatory activity. Although i.v. administration of IFN-gamma (10,000 U/mouse) strongly induced expression of IP-10 mRNA in the adherent cell population of the spleen, thioglycollate-elicited peritoneal macrophages were essentially unresponsive at the same dose. In contrast, D3 mRNA was expressed in both cell populations. This differential sensitivity of IP-10 mRNA expression was not restricted to stimulation by IFN-gamma as it was also seen when LPS (25 micrograms/mouse) was administered i.v. Expression of JE and KC mRNA, which encode cytokines related to IP-10, were also differentially expressed in elicited peritoneal macrophages from mice injected with LPS. Differential sensitivity was at least partially related to the state of macrophage activation because IP-10 mRNA was highly inducible in resident but not thioglycollate-elicited peritoneal macrophages. The eliciting agent was also an important determinant because proteose-peptone-elicited peritoneal macrophages were nearly as sensitive as splenic macrophages with respect to expression of IP-10 mRNA. IFN-gamma treatment induced IP-10 and D3 mRNA rapidly and transiently with the same time course in the spleen. IP-10 mRNA was not induced by IFN-gamma in TG-elicited macrophages regardless of the time after treatment. This differential expression of IP-10 was a consequence of different concentration requirements for IFN-gamma in the two cell types; thioglycollate-elicited macrophages required five- to 10-fold more IFN-gamma than did resident cells to achieve comparable IP-10 mRNA levels whether the agent was provided in vitro or in vivo. Thus variable sensitivity for induction of IP-10 mRNA was a characteristic of the macrophage itself and was not mediated by other cellular or molecular elements present in the inflammatory peritoneal cavity. The reduced sensitivity to IFN-gamma or LPS for expression of IP-10, JE, and KC mRNA as compared with TNF-alpha or D3 mRNA suggests that this distinct pattern of regulation may be restricted to members of these two related cytokine gene families that exhibit cell-type specific chemoattractant activity.     

The development of the resident pattern of endogenous peroxidatic activity in mouse peritoneal macrophages coincides with the expression of the differentiation antigen F4/80. A combined method for immunoperoxidase labeling and endogenous peroxidase cytochemistry

In the mouse the maturation of mononuclear phagocytes was followed by comparing the ultrastructural pattern of endogenous peroxidatic activity (PA) at different time points during an acute peritonitis induced with newborn calf serum (NCS). Exudate macrophages demonstrate PA only in lysosomes, whereas resident macrophages have reaction product in the nuclear envelope (NE) and rough endoplasmic reticulum (RER). Transitional cells called "exudate-resident" macrophages have PA in the NE, RER, and some virginal lysosomes. In addition, peroxidase-negative macrophages were also present. A monoclonal antibody, F4/80, that specifically recognizes a mouse macrophage differentiation antigen (Austyn and Gordon, 1981) was used in this study. To compare the indirect immunoperoxidase labeling of this antigen and the endogenous peroxidase cytochemistry on the cellular level, a combined method was developed. Finally, the method was applied to the peritoneal cells at different time points after intraperitoneal injection of NCS in mice. The relative numbers of cells demonstrating the different patterns of endogenous PA and the proportions of each subpopulation expressing F4/80 antigen were estimated. It appeared that the expression of the antigen F4/80 coincides with the development of the resident pattern of PA. It is therefore concluded that the macrophages with the resident pattern of endogenous peroxidase are derived from monocyte-like exudate macrophages. In addition, the results indicate that both exudate-resident macrophages and at least a part of the peroxidase-negative macrophages are transitional forms.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.     

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes

Summary BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 g/ml N. CWS for 3 days. NAPC from BALB/c mice given an i. p. injection with 100 g N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes. Abbreviations used: N. CWS, Nocardia rubra cell-wall skeleton; BRM, biological response modifier; MAF, macrophage activating factor; IL-1, interleukin 1; IL-2, interleukin 2; IFN-, interferon gamma; PCCM, peritoneal cell culture medium; SCCM, spleen cell culture medium; TCM, tumor culture medium; HI-FCS, heat-inactivated fetal calf serum; Con A, concanavalin A; PC, peritoneal cells; PEC, peritoneal exudate cells; WPC, whole peritoneal cells; APC, adherent peritoneal cells; TGC-APC, thioglycollateelicited adherent peritoneal cells; NAPC, nonadherent peritoneal cells; SN, supernatants; NK cells, natural killer cells; LAK cells, lymphokine activated killer cells E:T ratio, effector: target cell ratio; WSA, water soluble adjuvant; LPS, lipopolysaccharide; MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine     

Tissue distribution, structural characterization, and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity

Mac-3 is a mouse macrophage differentiation antigen defined by a rat anti-mouse monoclonal antibody (MAb),M3/84. The structure, biosynthesis, quantitative surface expression, and distribution of Mac-3 have been studied by radiolabeling and isolation with MAb-Sepharose, saturation binding, absorption, and immunofluorescence flow cytometry. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mac-3 migrates as a diffuse band with average Mr = 110,000. Labeling of intact cells with 125I and accessibility to MAb show it is present at least in part on the cell surface. Saturation labeling with 125I-MAb shows 4.2 X 10(4) cell surface sites on thioglycollate medium-elicited peritoneal macrophages. [35S]Methionine and [3H]glucosamine incorporation into Mac-3 by purified macrophages show it is a glycoprotein synthesized by these cells. Absorption shows Mac-3 is strongest in macrophages, present in lower quantities in lung, liver, bone marrow, and spleen, and undetectable in thymus, lymph node, brain, and heart. Immunofluorescent flow cytometry shows surface expression on thioglycollate-elicited macrophages but not bone marrow, spleen, lymph node, or thymus cell suspensions. Similar amounts of Mac-3 are immunoprecipitated from resident macrophages or macrophages elicited by sterile inflammatory agents, intracellular parasites, or immunomodulators, but the average Mr of Mac-3 varies from 92,000 to 110,000. Mac-3 is synthesized from precursor(s) of Mr = 74,000 and 79,000, identical in the different macrophages. Processing into the mature molecule, which migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a more diffuse band and varies in Mr among macrophage elicited by different agents and to a lesser degree between different preparations of the same type of macrophage, occurs in 15 to 30 min.     

Rat Macrophage C-Type Lectin Is an Activating Receptor Expressed by Phagocytic Cells

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both ¹²⁵I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20 % of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of ³H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton chain and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen     

Interleukin-4 is chemotactic for mouse macrophages.

An important component of the cell-mediated immune response often is the migration of macrophages to the site of immune activity. Although much evidence suggests that macrophage migration is regulated by antigen-specific T cells, the influence of T cell-derived cytokines on macrophage chemotaxis has not been well studied. Here we present evidence that interleukin-4 (IL-4), a cytokine derived from T helper 2 (Th 2) cells, is chemotactic for mouse peritoneal macrophages. In an in vitro chemotaxis assay using Boyden chambers, recombinant IL-4 was chemotactic for mouse peritoneal exudate macrophages. This response was inhibited in a dose-dependent manner by the anti-IL-4 antibody, 11B11. As shown here and previously, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), cytokines derived from T helper 1 cells, are not chemotactic for mouse macrophages.     

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen)

Galectin-3 (formerly called Mac-2 antigen) is a ∼30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the α-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate. Abbreviations: PBS, phosphate buffered saline; CNBR, cyanogen bromide; PMSF, phenyl methyl sulphonyl fluoride This revised version was published online in November 2006 with corrections to the Cover Date.