Modulation of macrophage fibronectin secretion by LPS.

The secretion of fibronectin (Fn) by rat peritoneal macrophages was found to be down-regulated by LPS. A sensitive ELISA was used to quantitate both substrate-attached and supernatant Fn. Thioglycollate-elicited peritoneal exudate cells were observed to release a considerable amount of Fn during the adherence procedure for macrophage purification. After this procedure, macrophage Fn levels peaked within 2 hr and then declined steadily to baseline levels by 96 hr. Fn release by exudate cells during adherence purification was less affected by cycloheximide treatment than was subsequent Fn secretion by purified macrophages. Macrophages elicited with thioglycollate and P. acnes displayed enhanced Fn secretory activity when compared with resident unstimulated cells. Exposure to lipopolysaccharide (LPS) suppressed the levels of immunoreactive Fn in supernatants of elicited cells. This inhibition was shown to be dose-dependent and most significant after 24 hr of incubation. The inclusion of polymyxin B in the culture medium did not reverse the LPS-induced inhibition of Fn production but did prevent LPS stimulation of interleukin-1 secretion in the macrophage cultures. These observations demonstrate that Fn secretion by macrophages is regulated according to the functional state of the cell.

Effect of fibronectin on antigen-induced lymphoproliferation and antibody synthesis in rats.

Macrophages are important positive and negative regulators of both primary and secondary antibody responses, and their activity may, in turn, be controlled by soluble mediators secreted by other cells. Fibronectin is a 440,000 dalton normal constituent of plasma and extracellular membranes that acts through macrophages to inhibit mitogen- and alloantigen-stimulated lymphoproliferation. We examined the effect of Fn on the antigen-stimulated lymphoproliferative and antibody responses in cells from trinitrophenol-derivitized keyhole limpet hemocyanin (TNP-KHL) primed rats. Fn in concentrations equivalent to normal plasma levels inhibited TNP-KLH-stimulated lymphoproliferation by unseparated lymph node leukocytes. When the experiment was repeated using purified lymph node T cells and added thioglycollate-induced peritoneal exudate macrophages or splenic adherent macrophages, Fn alone and TNP-KLH alone stimulated lymphoproliferation, but in combination they were strongly inhibitory. The effect was not due to decreased lymphocyte viability in the presence of both TNP-KLH and Fn. Nor was it due to complexes between TNP-KLH and Fn or to a simple alteration in the kinetics of lymphoproliferation. Fn had to be present with the TNP-KLH within the 1st hour of incubation. If macrophages were coincubated with TNP-KLH and Fn for 24 h, washed, and added to enriched T cells, inhibition was equivalent to that seen with continuous coculture. Similarly, coculture of TNP-KLH and Fn inhibited both total immunoglobulin and TNP-KLH-specific antibody synthesis at optimal concentrations of splenic adherent cells. However, at suboptimal levels of splenic macrophages, the combination was synergistic, stimulating more total immunoglobulin synthesis than either TNP-KLH or Fn alone. These data suggest that the inhibitory effect was dependent upon the concentration and phenotype of macrophages present in culture.

Stimulation of rat macrophage interleukin 1 secretion by plasma fibronectin.

Purified plasma fibronectin (Fn) enhanced the secretory activity of rat peritoneal exudate macrophages as measured by 35S-methionine incorporation into protein released into culture supernatants. Enhancement of protein secretion was dose-dependent and increased with time in culture. Addition of various concentrations of supernatant from cultures of macrophages with Fn resulted in a significant increase in thymocyte proliferation elicited by phytohaemagglutinin. The stimulatory activity of the supernatant was Fn dose-dependent and increased with increasing concentrations of macrophages. This thymocyte stimulatory effect was not due to the presence of Fn in the culture supernatant or to the minimal contamination with endotoxin detected in the Fn preparations. These data suggest that the inflammatory macrophage interaction with Fn results in the release of interleukin-1. They also are consistent with the reported ability of Fn to stimulate lymphocyte transformation.

Induction of lymphocyte blast transformation by purified fibronectin in vitro.

Purified rat plasma fibronectin (Fn) induces a dose-dependent, nonspecific proliferation of lymphoid cells isolated from spleen and lymph nodes, but has no effect on thymocytes. Proliferation of cells occurred after 4 to 5 days of incubation and was generally 5- to 10-fold greater than control cells cultured without Fn or in the presence of the same concentrations of rat serum albumin. The responding cell appears to be the T cell and requires the presence of adherent accessory cells (macrophages). Although purified T and B cells failed to demonstrate a significant increase in blastogenesis in the presence of Fn, reconstitution of T, but not B, cells with irradiated peritoneal exudate macrophages restored the stimulatory effect of Fn. Furthermore, comparison of the effect of various concentrations of macrophages on the restored T cell response shows that proliferative activity in the presence of Fn is dependent on a critical number of macrophages. Adding higher numbers of macrophages beyond the optimal concentration results in a sharp decline in lymphocyte responsiveness to Fn, although the proliferation of control cells continues to increase at these macrophage concentrations. Macrophages pulsed with Fn for 1 hr failed to evoke an increase in T cell responsiveness after 3 or 5 days of incubation. In addition, incubation of Fn-pulsed T cells with macrophages that had been precultured with or without Fn also failed to result in T cell activation. The effect of Fn on T lymphocyte transformation appears to be mediated indirectly through its interaction with the macrophage, because 125I-Fn, which shows significant binding to peritoneal exudate macrophages, fails to interact with purified T cells. Radiolabeled Fn also demonstrated little or no binding activity for unseparated lymph node cells.

Modulation of rat lymphocyte transformation by plasma fibronectin.

Purified rat plasma fibronectin (Fn) was studied for its ability to influence nonspecific lymphocyte transformation to various mitogens. Fn added to lymph node cells (LNC) stimulated with phytohemagglutinin (PHA), concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) resulted in a noncytotoxic dose-dependent inhibition of the blastogenic response. Effective inhibition (greater than 50%) of LNC responses to PHA and LPS occurred with concentrations of Fn that ranged from 10-50 micrograms/culture. The proliferative response to Con A was less affected by the presence of Fn: 50% inhibition was observed only with the highest concentration of Fn studied. Fn at low concentrations was more effective than Fn-depleted plasma in inhibiting lymphocyte responses to PHA. Optimal expression of the regulatory activity of Fn occurs during and following the peak proliferative period for both PHA and LPS responses. In order to evoke maximum inhibition it is necessary for Fn to be present within 12 hours following stimulation of LNC with mitogen. Inhibition does not appear to be the result of Fn-mitogen complexes which reduce the amount of mitogen available for stimulation, since increasing concentrations of either PHA or LPS did not significantly reduce the inhibitory effect. Furthermore, inhibition was not influenced by the concentration of fetal calf serum present in the culture medium. Thus, Fn may function as an important nonspecific immunoregulatory factor at inflammatory sites and in areas of tissue repair.

Heterogeneity, position, and functional capability of the macrophages in Peyer's patches.

Peyer's patches are thought to be deficient in macrophages capable of sustaining the induction of an immune response. The present study examined the position, morphology, and phagocytic activity of Peyer's-patch macrophages, and their ability to take up, in situ, a tracer antigen introduced through the intestinal lumen. Peyer's-patch macrophages were isolated in vitro, and nonspecific esterase activity of individual cells was quantitated. Macrophages concentrated within the epithelium overlying Peyer's patches and in the subepithelial zone showed phagocytic activity and the ability to take up exogenous ferritin. Adherent macrophages demonstrated heterogeneity of esterase activity, with approximatley 15% having none. However, esterase activity in macrophages from lymph nodes was equally heterogeneous. The profile of esterase activity of Peyer's-patch macrophages seemed intermediate between those of macrophages from submandibular and mesenteric nodes. Tingible body macrophages were concentrated in germinal centers and were poorly adherent. The results of this study indicate that macrophages similar to those in lymph nodes are concentrated in the region where they may intercept antigens entering through overlying specialized epithelium. The presence of plasma cells in the same region suggests that local reaction to antigens, perhaps modulating further antigen entry, is supported by these macrophages. Perhaps the proportion of functional macrophages in the entire Peyer's patch is too low to support immune induction in in vitro-tests.

Lymphoid cell interactions in allograft immunity. Failure of hamsters to generate suppressor cells upon specific immunization.

The alloantigenic reactivity of hamster spleen cells from unsensitized animals was analyzed in vitro. When compared with normal MHA cells, MHA anti-CB spleen cells responded better or to the same degree in mixed lymphocyte reaction (MLR) during the first 3 days after alloantigenic exposure. However, by day 5 postsensitization there was a signigicant reduction in spleen cell MLR activity, which returned to normal or even an exaggerated response 7 days following immunization. We examined the possibility that the reduction in MLR responsiveness 5 days following immunization might be attributable to the generation splenic suppressor cells. Irradiated spleen cells from unsensitized and from MHA anti-CB donors were cocultured with normal responder MHA lymph node cells stimulated by (MHA x CB)F1 hybrid lymph node cells in MLR. No regulatory effect of either cell population was seen, indicating that even hamster spleen cells from alloimmune donors fail to suppress the mitotic response of normal lymph node cells to allogeneic targets.