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The expression of galectin-3 (formerly known as IgE-binding protein or Mac-2) in rat bone marrow (BM) was investigated by FACS, immunocytochemical and immunoblot analysis. The functional significance of rat recombinant galectin-3 on mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven proliferation of macrophage progenitors and gene transcription was further examined. Immunocytochemical analysis of in situ BM sections demonstrated galectin-3 in myelopoietic cells and surrounding stroma, whereas erythropoietic and lymphopoietic environments essentially lacked galectin-3 expression. FACS analysis demonstrated that incubation of freshly isolated BMC with lactose, a competing ligand for galectin-3 binding to glycoconjugates, decreased binding of antigalectin antibodies to cells primarily expressing the myeloid antigen recognized by mAb His-54. Similarly, lectin-mediated binding of exogenous galectin-3 to myeloid lineage cells was also demonstrated. Immunoblot analysis of BM eluates demonstrated galectin-3 both in the extracellular matrix and in a lactose elutable form, bound to the surface of BMC. [3H]Thymidine incorporation studies on BMC cultured in the presence of galectin-3 demonstrated suppression of GM-CSF-induced proliferation by galectin-3. In addition, differential display analysis of immediate early gene expression in BMC cultured in the presence of galectin-3 revealed a 76.2% inhibition of GM-CSF-induced gene transcription by galectin-3 assessed by the number of PCR-fragments generated. Our data suggest a role for galectin-3 in the organization of myelopoietic compartments in rat BM and regulation of the action of growth factors on myelopoietic precursor cells.

Human alveolar macrophages (AM) from thirteen patients, who were suffering from various lung diseases were harvested by bronchoalveolar lavage. Peripheral blood monocytes from eight healthy donors were isolated by Ficoll-Hypaque gradient centrifugation and adherence to plastic surface. To detect the VEP13 antigen (CD16) on these cells, a rosette assay employing ox erythrocytes coated by the CrCl 3 method with purified VEP13 monoclonal antibody (E o -VEP13) was used. A mean of 31.3 % of freshly isolated AM and 3.9 % of blood monocytes formed E o -VEP13 rosettes. Monocytes cultured for 3 or 6 days in the presence of a supernatant from mouse L929 cells, which had been shown previously to improve long-term viability of human monocytes in culture, showed 12.5 % and 25.3 % E o -VEP13 rosettes, respectively. No significant increase in VEP13 antigen expression was noted by culturing monocytes without L929 cell supernatant. The factor in L929 supernatant that induces VEP13 antigen expression has not been identified. Tunicamycin at 10 υg/ml inhibited significantly VEP13 antigen expression on monocytes. In contrast, IgG rosette formation was not reduced by tunicamycin. Our data show that subpopulations of native human AM and peripheral blood monocytes cultured in presence of a supernatant of L929 fibroblasts containing mainly murine CSF may express the CD16 antigen, which is normally found on large granular lymphocytes (LGL). Suppression by tunicamycin indicates that Fc receptor glycosylation takes place during a later differentiation step of mononuclear phagocytes.

In this study the effect of alveolar (AC) or peritoneal (PC) lavage cells from Lewis rats on the mitogen induced proliferation of syngeneic spleen cells was investigated. When total spleen cells were stimulated with PHA (5 μg/ml) or ConA (6 μg/ml), the addition of AC or PC and of their adherent (A) or non-adherent (NA) fractions had dose dependent suppressive effects. After passing the spleen cells through a Sephadex G-10 column, the eluted cells responded poorly to the mitogens. Addition of low numbers (2x10 3 = 10%) AC or NA-AC improved this response, whereas higher concentrations (5 or 10 %) of these cells or any concentration of A-AC, PC, A-PC or NA-PC showed either none or a suppressive effect. The cells which strongly suppress mitogen induced lymphocyte proliferation have macrophage morphology, are adherent, esterase positive, bear Fc receptor for IgG and bind ganglioside coated erythrocytes. NA-AC were further separated into Ia-depleted (Ia - ) and Ia-enriched (Ia + ) population. The Ia + fraction contained 80 to 90% Ia bearing cells, supported the mitogen induced stimulation of Sephadex G-10 depleted spleen cells and was ineffective in suppression. Interestingly, these cells did not bind ganglioside treated sheep erythrocytes. On the other hand, the Ia-negative NA-AC fraction and particularly the A-AC population strongly expressed the «ganglioside receptor» as well as typical macrophage markers like Fc receptors, latex-phagocytosis and non-specific esterase. Therefore, the «ganglioside receptor» may be a useful marker to define suppressor macrophages.

Rat alveolar and peritoneal macrophages were tested in a rosetting assay for their capacity to bind rabbit IgG antibody sensitized sheep erythrocytes (EA) before and after various times of incubation with proteolytic enzymes. With most enzymes, a biphasic effect on EA rosette formation was observed: an initial enhancement was followed by a reduction of the number of rosette-forming cells (RFC). The extent and rate of these changes depended on the type and concentration of the enzyme and on the amount of IgG antibodies used to sensitize the sheep erythrocytes. At low enzyme concentrations and with lowly sensitized EA, the phase of RFC enhancement was more prominent and prolonged. With an increase of enzyme concentration, the rate of reduction of RFC was increased. With regard to different enzymes, the highest rate of receptor degradation was found with pronase, followed by trypsin, whereas α-chymotrypsin had little receptor-degrading activity. A slight enhancement not followed by a loss of EA-rosetting activity was induced by granulocyte elastase at the concentrations studied. The results may give an explanation to contradicting reports in the literature, in which a single dose or just a few doses of proteolytic enzymes were employed and no kinetic studies were performed. In addition, EA-rosette-inhibiting material was found in supernatants of macrophage cultures, suggesting that receptor-like material was shed during incubation in serum-free medium. This material lost its EA-rosette-inhibiting capacity after proteinase treatment.

Anti-macrophage sera (AMS) produced in rabbits against rat alveolar (AM) and peritonealmacrophages (PM) were tested for their selective capacity to kill 51 Cr-labelled AM, PM and thymus lymphocytes (TL) of rats. All non-absorbed AMS were able to kill both types of macrophages as well as TL in thepresence of complement. However, the cytotoxic titre (CT) and the percent of specific 51 Cr-release was always higher on homologous macrophages (as used for immunization) than on heterologous macrophages (of the other population). On both types of macrophages it was stronger than on TL. After absorptions of AMS with insolubilized rat plasma and fetal calf serum (FCS), with raterythrocytes and with non-adherent cells from rat kidney, thymus, spleen and bone marrow (PEKL-absorptions) all AMS reacted only with macrophages and not with TL. When these antisera were absorbed repeatedly with limited amounts of AM or PM afunctional specificity was observed for the cell type which was not used for absorption. Further absorptions of these AMS with the same cells removed completely all cytotoxic activity for both macrophage populations. It is supposed that AM and PM have at least two different antigenic determinants present ontheir surface but their density is different in the two cell populations. Antigens specific for one cell type could not be detected in complement dependent cytotoxicity.