On the mechanism of binding of human lymphocyte products to guinea pig macrophages

When lymphocytes from a majority of patients with cancer are incubated with encephalitogenic factor, a lymphocyte product is released that reduces the anodic electrophoretic mobilities of guinea pig macrophages and fixed, tanned sheep erythrocytes. Although these reactions are not specific for cancer, it is distinctly possible that in patients with cancer, products from stimulated lymphocytes are capable of altering the surfaces of the patients' own macrophages, thereby modifying the course of their disease. In this paper, we attempt to elucidate some mechanisms for the binding of lymphocyte products to macrophages, such as occurs in the macrophage electrophoretic mobility (MEM) test, since this may be of general interest. Binding of lymphocyte product to macrophages has been monitored by measurements of their electrophoretic mobilities and by electron microscopic determination of the density of binding of electron-dense, cationic colloidal iron hydroxide particles to their surfaces. The results show that the lymphocyte products reduce the net surface negativity of the macrophages by (coulombic) binding of this net positively charged material to sialic acids at the macrophage surface. Product-binding can be prevented by prior treatment of the macrophages with neuraminidase. It appears that only a minority of sialic acids are involved in the binding process, which occurs without demonstrable blocking of adjacent sialic acids or redistribution of such sites over the macrophage surface. Parallel experiments with fixed tanned erythrocytes also suggest that binding of lymphocyte products is not solely determined by surface sialic acids, although it cannot occur without them.     

Hapten binding assay using guinea pig macrophages

An assay for testing the binding of haptens to antigen-presenting cells was developed. The test consists of consecutive incubation of macrophages with the tested hapten, followed by incubation with a radioactively labeled standard. If the first hapten occupies a certain number of binding sites, the other one (radioactively labeled standard) reveals reduced binding. The binding assay in the present modification is fully suitable for measuring the binding of water-soluble haptens. It was found that from a panel of tested haptens, DNFB, beta-bromostyrol and phenylacetaldehyde exhibit strong binding.     

Bombesin in human and guinea pig alveolar macrophages

Bombesin found in neuroepithelial bodies and oat cell carcinoma of the lung, is thought to play an important role in normally developing and malignant lung. Monocytes-macrophages and human small cell lung carcinoma cells share several features, including macrophage-specific surface markers and the expression of functional receptors for bombesin-like neuropeptides and growth factors. Because small cell lung carcinoma cells synthesize immunoreactive bombesin, we investigated the possibility that alveolar macrophages also contain bombesin, a plausible hypothesis considering the many reports of neuropeptide production by immune cells and cells of bone marrow origin. Adherent human peripheral blood mononuclear cells as well as human and guinea pig alveolar macrophages were found to contain bombesin. The peptide was detected by radioimmunoassay, immunohistochemistry, high-pressure liquid chromatography with the use of different monospecific antibodies.     

Macrophage-lymphocyte interaction: antigen-independent binding of guinea pig lymph node lymphocytes by macrophages.

The antigen independent binding of guinea pig lymph node lymphocytes by glass-adherent macrophages was investigated. Binding was found to be mediated by a trypsin digestible, divalent cation-dependent, temperature-sensitive macrophage receptor mechanism that was not competitively inhibited by excess immunoglobulin. Data are presented to indicate that in the absence of antigen, macrophages were capable of binding both thymus-derived and bone marrow-derived lymphocytes without apparent selectivity, and further, that the binding of neither cell was mediated by surface membrane-associated immunoglobulin.     

Effect of glycosylation inhibitors on binding properties of the Fc gamma receptors from guinea pig peritoneal macrophages.

Guinea pig peritoneal macrophages possess on their surfaces receptors for the Fc region of IgG immunoglobulins (Fc gamma R). The cells treated with the glycosylation inhibitors tunicamycin or monensin interacted with IgG with a higher affinity and showed a lower number of IgG-binding sites in comparison with control cells. This indicates that the interaction of IgG with the macrophage Fc gamma receptor depends on the degree of glycosylation of the cell surface glycoconjugates and that glycosylation of the macrophage Fc gamma receptor is important for the expression of the mature form of the receptor.     

In vitro killing of tumor cells by soluble products of activated guinea pig peritoneal macrophages

A partially purified fraction from supernatants of macrophage cultures from BCG-infected guinea pigs, which is rich in anti-Listeria activity, was cytotoxic to malignant and not normal cells. The susceptibility of target cells to this material varied considerably. Concentrations of egg white lysozyme, equal to those of the macrophage product, had considerably less effect on line 1 hepatoma cells.     

Substance P: binding properties and studies on cellular responses in guinea pig macrophages

The neuropeptide Substance P (SP) has been recognized to modulate functional activities of inflammatory cells. We have previously shown that it mediates macrophage activation. In this study we examined binding characteristics of SP and searched for additional evidence of heightened metabolic activity of guinea pig peritoneal macrophages upon challenge with this peptide. Radioligand studies indicated the existence of a homogeneous class of specific binding sites with high affinity for SP on macrophages. Scatchard analysis yielded an apparent KD of 1.9 +/- 0.4 X 10(-8) M (range: 1.4 to 2.4 X 10(-8) M), which was confirmed by kinetic studies. Binding was dose related, saturable, reversible, and could be inhibited by the SP antagonist (D-Pro2,D-Phe7,D-Trp9)-SP. Examination of peptide structural requirements revealed that both the COOH- and NH2-terminus contribute to receptor-ligand interaction. Other members of the tachykinin group of peptides were devoid of stimulatory action on macrophages. Cellular responses after engagement of the receptor sites by SP included downregulation of the membrane-associated enzyme 5'-nucleotidase and stimulation of synthesis and release of arachidonic acid metabolites, as well as of the lysosomal enzyme ADGase. These actions were specific as evidenced by immunoabsorption experiments. Our findings demonstrate that macrophage activation afforded by SP is effected through a receptor-mediated mechanism. Liberation of proinflammatory and immunomodulating substances in response to SP may be relevant to the pathogenesis of neuroinflammatory disease.     

Opiate binding to subcellular fractions from guinea pig ileum

Binding of 3H-etorphine and 3H-D-Ala2-D-Leu5-enkephalin to opiate receptors in synaptosomal and microsomal fractions prepared from guinea pig ileum homogenates has been studied. It is found that the dissociation constants for etorphine from all fractions are the same. The binding capacity for etorphine for the purified synaptosomal fraction is greater than for other fractions by a factor of 5. For the enkephalin derivative binding to the microsomal fraction the dissociation constant is greater than for etorphine while the binding capacity is a factor of 3 lower. These results are in contrast to the case for binding to central nervous system subcellular fractions.     

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig

The majority of glycosaminoglycans synthezied in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment, indicating that the glycosaminoglycanas existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine, Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicate that ΔDi-diS_E derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [³⁵S]sulfate and [³H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the relese of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.