Expression of endothelial cell IgG Fc receptors and markers on various cultures.

OBJECTIVE: To determine and compare the expression of endothelial cell IgG Fc receptors (Fc gamma R) and markers on various kinds of cultures. METHODS: Human breast microvascular endothelial cells (HMVEC), human aortic endothelial cells (HAEC), human umbilical vein endothelial cells (HUVEC) and canine aortic endothelial cells (CAEC) were stimulated with cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The binding of anti-Fc gamma receptor (Fc gamma R) type I, II and III antibodies was measured using an enzyme-linked immunosorbent assay (ELISA). The constitutive expression of endothelial cell markers was examined using anti-von Willebrand factor antibodies, Dil-low density lipoprotein (Dil-Ac-LDL) and fluorescein isothiocyanate (FITC)-labeled ulex europaeus agglutinin-1. RESULTS: The binding of anti-Fc gamma R II was significantly increased by the simultaneous stimulation with TNF-alpha and IFN-gamma on all three types of human endothelial cells (ECs), but not on canine endothelial cells. Enhanced Fc gamma R II expression was most significant when human ECs were cultured in endothelial cell basal medium (ECBM). However, the expression of Fc gamma R II on CAECs could not be induced by human cytokines even after they were cultured in ECBM for 3 passages. Endothelial cells also showed diversity for the constitutive expression of classic markers. CONCLUSIONS: This study demonstrate that cytokines TNF-alpha and IFN-gamma enhance low-affinity Fc gamma R expression on human endothelial cells in vitro. The results indicate that heterogeneity of endothelial cells exists not only on constitutive expression but also on stimulative expression.

Detection of Fcgamma receptors on human endothelial cells stimulated with cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma).

This investigation was conducted to detect Fcgamma receptors (FcgammaR) on cytokine-stimulated human endothelial cells (EC) by measuring anti-FcgammaR MoAb binding with an ELISA. TNF-alpha and IFN-gamma significantly increased the expression of FcgammaR type II (FcgammaRII) and type III (FcgammaRIII) on aortic EC. Simultaneous treatment with both cytokines had a synergistic effect and pretreatment of EC with IFN-gamma augmented the effect of TNF-alpha. The greatest effect was the increase (up to four-to-six-fold) in expression of FcgammaRII found by the simultaneous treatment of aortic EC with both cytokines. The receptors were expressed on the cell surface and showed receptor capping after incubation at 37 degrees C. This study showed that the inflammatory cytokines TNF-alpha and IFN-gamma enhanced low-affinity FcgammaR expression on human EC in vitro. The expression of FcgammaR may contribute to the specific localization of circulating immune complexes on blood vessels in areas of vasculitis.

Use of peroxidase:anti-peroxidase immune complexes as markers for Fc gamma receptors in vitro.

In species for which monoclonal antibodies are not yet available, the demonstration of Fc receptors has relied on a variety of ligand-based assays including the binding of antibody-coated erythrocytes, radiolabeled monomeric immunoglobulin, and non-physiologic aggregates of immunoglobulin. In order to study the binding of small immune complexes to Fc receptors on canine monocytes, a new method was developed using an enzyme-linked immune complex. The ability of rabbit polyclonal peroxidase:anti-peroxidase (PAP) immune complexes to bind to freshly isolated canine peripheral blood monocytes was characterized using standard ELISA techniques. The binding of rabbit polyclonal PAP to monocytes was time and concentration dependent and reversible. This binding was saturable with increasing concentrations of PAP and could be blocked by soluble rabbit IgG or rabbit Fc fragments. The blocking and saturation curves for canine monocytes were suggestive of multiple classes of Fc gamma binding sites. In contrast to intact PAP complexes, the binding of F(ab) PAP preparations or free horseradish peroxidase was minimal. The use of commercially available PAP preparations provides a reproducible, inexpensive, and non-radioactive measure of Fc gamma receptor binding on canine cells. In addition, these findings suggest caution in using heterologous PAP as a histochemical reagent in tissues expressing Fc gamma receptors.

Plasmid profiles and resistance to antimicrobial agents among Salmonella enteritidis isolates from human beings and poultry in the midwestern United States.

In the study reported here, 121 Salmonella enteritidis isolates from human beings and 467 isolates from nonhuman sources were analyzed for plasmid pattern and susceptibility to a panel of antimicrobial agents commonly used as biologic markers. A significant (P < 0.05) number of isolates from nonhuman sources were resistant to beta-lactam antibiotics and tetracycline. Resistance to aminoglycosides, quinolones, and trimethoprim/sulfamethoxazole was uncommon. Of the 588 isolates, 445 (76%) were resistant to 2 or more antimicrobial agents. Sixty of 121 (50%) S enteritidis isolates from human beings were susceptible to all 12 antimicrobial agents, but 425 of 467 (91%) S enteritidis isolates from nonhuman sources expressed resistance to 1 or more of the antimicrobial agents used in the study. Analysis of plasmid profiles revealed that significantly (P < 0.05) more isolates from nonhuman sources had high molecular weight plasmids than did isolates from human beings. Isolates from ceca of chickens were associated with patterns of low molecular weight plasmids. Analysis of results of the study revealed similarities among S enteritidis from human beings and eggs, as determined on the basis of plasmid profiles and antibiotic susceptibility patterns, which may implicate eggs as one of the potential sources for infection of human beings. In addition, periodic monitoring of a substantial number of Salmonella isolates to detect drug resistance may be a prudent practice for use in revising the list of antimicrobial agents commonly used in human beings and other animals.

In vivo and in vitro characteristics of interleukin 6-transfected B16 melanoma cells.

Interleukin 6 (IL-6) is a multifunctional cytokine important in the inflammatory response. Its potential role as an antitumor agent has been suggested by its demonstrated activity in a variety of tumor models. The mechanism of antitumor activity has been proposed to be its enhancement of cytotoxic T-cell function. In the current work we demonstrate clear antitumor activity for this cytokine in a nonimmunogenic tumor system. B16 melanoma cells transfected with the human IL-6 complementary DNA demonstrated slower tumor growth in vivo. Tumors that developed from these cells had a prominent stromal matrix, an easily recognized infiltration of inflammatory cells, fewer mitotic figures, and fewer blood vessels. These in vivo findings corresponded with a greater adhesion of the IL-6-transfected B16 cells to stromal matrix proteins (laminin, fibronectin, and vitronectin) and a less prominent vascular response in an intradermal angiogenesis assay. Therefore, we propose that with weakly antigenic tumors, such as B16 melanoma, IL-6 may mediate important antitumor responses by nonspecific proinflammatory mechanisms.

Effect of host age on tumor-associated angiogenesis in mice.

Previous reports on the slower growth of tumors in senescent mice have suggested a decrease in tumor angiogenesis in these animals, but such an observation has not yet been documented quantitatively. In this study, we report the relative amount of tumor angiogenesis and tumor volume for two different types of tumor in 11 young (8-9-wk old) versus nine older (19-mo old) male C57BL/10 mice. B16 melanoma or SP1 methylcholanthrene-induced fibrosarcoma cells were injected into the ventral skin of mice. After 3 days, the mice were killed and the injection sites were examined for angiogenesis surrounding the tumor (centrally directed tumor angiogenesis), nerve-associated angiogenesis, and tumor volume. In the older mice, there was significantly less centrally directed tumor angiogenesis for both tumors tested, and nerve-associated angiogenesis was decreased for B16 melanoma. The mean tumor volume for the B16 implants was smaller for the older animals, but the mean SP1 tumor volumes were identical for both age groups. These findings support the hypothesis that tumor growth in older animals is associated with less formation of new blood vessels, and this may explain the slower tumor growth observed in aged animals with certain experimental tumors.

Investigation of tumor angiogenesis in an id mouse model: role of host-tumor interactions.

The role of host-tumor interactions in tumor angiogenesis was studied in an id mouse model. Although many previous studies have described the effects of certain host factors (age, nutritional status, tumor location, etc.) on experimental tumor systems, the effects of these factors on tumor angiogenesis have not been reported. In preliminary studies we inoculated cloned tumor cells id into syngeneic mice and found positional differences in tumor growth that did not correlate with measured angiogenesis levels. In addition, while tumor growth was inhibited by radiation, tumor-induced (but not fibroblast-induced) angiogenesis was not. This id system may provide an assay not only for examining the effect of host factors on tumor angiogenesis, but also for elucidation of the mechanisms underlying neovascular induction and host response.

Lymphocyte-induced angiogenesis factor is produced by L3T4+ murine T lymphocytes, and its production declines with age.

Lymphocyte-induced angiogenesis factor (LIA) is a product of T lymphocytes which has been shown to stimulate new vessel formation. Because immune senescence most profoundly affects T lymphocyte functions, we suspected that LIA production would decline with age. An assay for angiogenesis stimulated by allogeneic reaction was performed by injecting spleen cells from young or old donor mice into the skin of irradiated allogeneic recipient mice. The spleen cells from young mice induced a significantly greater number of vessels than did cells from older mice. In additional experiments, spleen cells from young and old animals were treated with a monoclonal antibody GK 1.5) directed at the L3T4 antigen on murine T helper lymphocytes. Such treatment significantly reduced the new vessel formation induced by young lymphocytes but had no effect on that induced by lymphocytes from old animals. Studies employing indirect immunofluorescence demonstrated that the proportion of L3T4+ cells in the mononuclear fraction of splenocytes was nearly identical in both young and old mice. From these investigations we can conclude that (1) L3T4+ lymphocytes are responsible for LIA production, and (2) production, like that of other T lymphokines, declines with age.

Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response.

Rat PMN isolated from peripheral blood show a small amount of high-affinity (specific) binding of [3H]-LTB4 at nanomolar concentrations. This binding is reversible and has a stereospecificity similar to rat PMN aggregation in response to several LTB4 analogs. This population of binding sites shares many characteristics with a population of high-affinity binding sites in human PMN; however, human PMN bind a significantly greater amount of [3H]-LTB4 to a second population of specific binding sites that is not present in rat PMN. The aggregation responses of human and rat peripheral blood PMN to LTB4 are similar in magnitude and specificity, but unlike human PMN, LTB4 fails to elicit a chemotactic response in rat PMN at concentrations from 10(-10) M to 10(-6) M. Rat PMN also fail to metabolize exogenous LTB4 when compared with human PMN. These data suggest that different PMN functions, such as chemotaxis and aggregation, may involve different classes of specific receptors. The finding that rat PMN do not exhibit chemotaxis to LTB4 calls for a reevaluation of the relevance to inflammation in humans of studies of inflammation performed in rat models.

Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes.

In this paper we have described the binding of nanomoler concentrations of [3H]leukotriene B4 (LTB4) to human polymorphonuclear leukocytes. Because up to 80% of the total [3H]LTB4 binding was blocked by excess (greater than 100 times) [14C]LTB4, the majority of binding is specific. Stereospecificity of the LTB4 binding is demonstrated by the diminished relative abilities of the 6-trans-and 12-epi-6-trans- isomers of LTB4 to block [3H]LTB4 binding. With these two isomers 3-10-fold higher than [14C]LTB4 concentrations were needed for equivalent inhibition of [3H]LTB4 binding. This difference is quantitatively less dramatic than the differences between these isomers in many in vitro functional assays such as chemokinesis, chemotaxis, and degranulation. Binding of [3H]FMLP is not blocked at greater than 100-fold excess of LTB4. The binding of [3H]LTB4 to cells appears to be essentially irreversible at 4 degrees C, but not at 37 degrees C where initially bound LTB4 is rapidly converted to metabolites which then enter the medium. These results suggest the presence of a saturable, stereospecific site for LTB4 on PMN. The association of LTB4 binding and the initiation of pharmacological responses to LTB4 will require further studies.