Robert J. Walter

Steroid Sex Hormones and Macrophage Function: Modulation of Reactive Oxygen Intermediates and Nitrite Release

PROBLEM: In general, females have a more active immune response than do males. The effects of female sex hormones on lymphocytes have been studied extensively but their effects on macrophages are poorly understood.

Direct visualization of formylpeptide receptor binding on rounded and polarized human neutrophils: cellular and receptor heterogeneity.

We have used light microscope autoradiography to visualize binding of the formylhexa‐peptide, N‐formyl‐norleucyl‐leucyl‐phenylalanyl‐norleucyl‐(125l)tyrosyl‐lysine to rounded and spontaneously polarized human polymorphonuclear leukocytes. These cells possess receptors known to bind with high specificity and great avidity to the chemotactic formylpeptides. Cells adherent to glass slides were exposed to (125I)‐hexapeptide at 4°, fixed, and autoradiographed. Hexapeptide binding was studied over the biologically active range of peptide concentrations varying from 0.63 nM to 10 nM and autoradiographic silver grains counted on 200 rounded or 50 polarized cells at each concentration. Examination of histograms plotted from these data revealed for rounded cells: 1) two major peaks at each concentration indicating the existence of two neutrophil subpopulations, the predominant subpopulation binding one‐half as much formylpeptide (peak I) as the other (peak II); 2) progressively increasing proportions of cells in peak II as the free hexapeptide concentration increased. Accordingly, at 0.63 nM hexapeptide, peak II comprised only 8% of the total cell number, whereas at 10 nM, this peak represented 35% of the total cells. This suggested that different types of receptors may exist in the two cell subpopulations (high/low affinity or high/low negative cooperativity) and that these receptor types were expressed differentially on these subpopulations. Thus, cellular heterogeneity within the neutrophil population and receptor heterogeneity among hexapeptide receptors on an individual cell were both observed here. Each of these may significantly affect neutrophil functional responses to the chemotactic formylpeptides and may explain, at least in part, the curvilinearity in the Scatchard plots of formylpeptide receptor binding that has recently been reported.

Localization of chemotactic peptide receptors on rabbit neutrophils

The chemotaxis of blood leukocytes is initiated by the binding of a chemoattractant to specific receptors on the leukocyte cell surface. Although a great deal is known about the biochemical and morphological events accompanying chemotactic activation, there is very little morphological information about the chemoattractant receptors themselves. This latter information is needed so that we may understand the mechanism by which these inflammatory cells detect and respond to chemical gradients. One class of chemotactic factors extensively used to characterize the complex behavioral responses following leukocyte activation are the synthetic formylmethionyl peptides. These peptides, now known to be the analogs of the naturally occurring N-terminal peptides produced by bacteria, are released into culture medium and are believed to be responsible, at least in part, for the accumulation of leukocytes at the sites of bacterial infection. We have localized the receptors for the chemotactic hexapeptide N-formylnorleucyl-leucyl-phenylalanine-norleucyl-[125I]tyrosyl-lys ine [N-fNle-Leu-Phe-Nle-[125I]Tyr-Lys] on whole rabbit peritoneal neutrophils (PMN) using light microscope autoradiography. By this method, the inherent formylpeptide receptor distribution on cells incubated at 4 degrees C appears to be uniform over the surface of both rounded and structurally polarized PMN. Following a short 37 degrees C incubation, cells retain a large proportion of labelled hexapeptide at or near the cell surface and, in addition, polarized PMN redistribute the hexapeptide anteriorly away from the cell uropod.

In vivo effects of recombinant interferon-γ : augmentation of endotoxin-induced necrosis of tumors and priming of macrophages for tumor necrosis factor-α production

Abstract Recombinant interferon-γ (rIFN-γ) is currently undergoing clinical trials in cancer patients. In this paper, we assessed the in vivo antitumor effects of this lymphokine in rodents. Recombinant murine IFN-γ or control medium was injected intraperitoneally for 5 days into mice with subcutaneous Meth A tumors. An injection of a suboptimal dose of endotoxin (2 μg) on the fifth treatment day caused significant necrosis of tumors in the IFN-γ-treated group while causing essentially no necrosis of tumors in the control group. Next, we examined macrophages isolated from rats treated for 9 days with either IFN-γ or saline. Endotoxin stimulated release of significantly higher amounts of TNF-α from macrophages from the IFN-γ-treated group compared to macrophages from the control group. A polyclonal antiserum against recombinant murine TNF-α abrogated all of the TNF cytotoxic activity from these rat macrophage supernatants, while control rabbit serum had no effect. These results provide strong evidence that rIFN-γ can prime macrophages in vivo for TNF-α synthesis.

Defects in monocyte chemotaxis in patients with neoplastic disease

The immuno-surveillance system is generally believed to detect and deter the spread of neoplastic disease. As major effecters of this complex system, infiltrating macrophages or monocytes accumulate at tumor sites and are thought to participate directly in tumor destruction [26, 381. Macrophage migration plays an essential role in such cell-mediated immune responses and in inflammatory reactions [65]. Studies of experimental tumor models have shown that macrophage migration is reduced in tumor-bearing animals [ 34, 41, 481. The migration of macrophages into sites of inflammation and macrophage accumulation around tumors are mediated primarily by soluble and surface-adherent chemical stimuli [40,60]. Soluble factors have been used for in vitro assays of directional locomotion, i.e., chemotaxis, to evaluate leukocyte function in tumor patients. Chemotaxis assays have employed primarily lymphokines (lymphocyte derived chemotactic factors) or complement fragments (C5a) as attractants for human monocytes [9, 44, 601. In addition, we have recently used a third group of chemoattractants, the synthetic formylpeptides, to study the migration of tumor patient neutrophils (PMN) and monocytes [68]. Chemotaxis of PMN from tumor patients is generally normal but a deficiency is clearly evident in the chemotaxis of monocytes from patients with malignant disease. In this review, we discuss the methodology used for leukocyte chemotaxis studies, summarize the data currently available on the chemotaxis of tumor patient leukocytes, and assess the biological and clinical significance of the leukotactic defect seen in these patients.

Increased viability and differentiation of normal and dystrophic striated muscle in vitro.

SummaryPrimary cultures of muscle from normal (line 412) and dystrophic (line 413) chick embryos were exposed to corticosterone-21-acetate (C-21-A) or sodium ibuprofen (Motrin) for 28 d after myotube formation. Ibuprofen (0.5 to 500 μg/ml) or C-21-A (0.4 to 40 μg/ml)-treated cultures were fixed and assessed semiquantitatively using phase microscopy. On this basis, ibuprofen (50 μg/ml) and C-21-A (40 μg/ml) seemed to be effective in maintaining both normal and dystrophic muscle cultures. Using ibuprofen and C-21-A at these concentrations, experiments were repeated and analyzed quantitatively. Ibuprofen maintained culture viability (up to 68% more myotubes than untreated controls) but had no significant effect on the number of striated cells. C-21-A effectively maintained culture viability (up to 73% increase) and strongly promoted the formation of striated cells in these cultures (up to a sixfold increase). Both normal and dystrophic cultures were affected similarly by these agents, but the dystrophic cultures showed more consistent if not more extensive improvements in the parameters examined here. Thus, it seems that ibuprofen and C-21-A may affect both normal and dystrophic muscle directly to maintain survival and even promote differentiation.