Modulation of neutrophil function by the tripeptide feG

Abstract

Background: Neutrophils are critical in the defense against potentially harmful microorganisms, but their excessive and inappropriate activation can contribute significantly to tissue damage and a worsening pathology. Through the release of endocrine factors submandibular glands contribute to achieving a balance in neutrophil function by modulating the state of activation and migratory potential of circulating neutrophils. A putative hormonal candidate for these effects on neutrophils was identified as a heptapeptide named submandibular gland peptide T (SGP-T; sequence = TDIFEGG). Since the tripeptide FEG, derived from SGP-T, and its D-amino acid analogue feG had similar inhibitory effects on inflammatory reactions, we investigated the effects of feG on human and rat neutrophil function.

Results: With human neutrophils feG had no discernible effect on oxidative burst or phagocytosis, but in picomolar amounts it reduced PAF-induced neutrophil movement and adhesion, and the binding of CD11b by 34% and that of CD16b close to control values. In the rat feG (10-11M) reduced the binding of CD11b and CD16 antibodies to PAF-stimulated circulating neutrophils by 35% and 43%, respectively, and at 100 micrograms/kilograms intraperitoneally feG reduced neutrophil in vivo migration by 40%. With ovalbumin-sensitized rats that were challenged with antigen, feG inhibited binding of antibodies against CD16b but not CD11b, on peritoneal leukocytes.

Conclusions: The inhibitory effect of feG on neutrophil movement may be mediated by alterations in the co-stimulatory molecules CD11b and CD16.

Figure 1

feG and neutrophil migration. The dose response relationship for feG inhibition of PAF (10^-9M) induced neutrophil migration. (mean ± SEM; n = 3–7). Statistically significant differences (*P < 0.05) between the indicated values and the inhibition seen in absence of peptide.

Figure 2

feG and neutrophil adhesion. Effects of feG on neutrophil adhesion. A. feG did not affect the basal adhesion of neutrophils to gelatin. B. At doses of 10^-11M and 10^-10M feG inhibited neutrophil adhesion to gelatin induced by 10^-9M PAF. (mean ± SEM; n = 5). Statistically significant differences (P < 0.05): # greater than Control in panel (A); * less than no peptide.

Figure 3

feG and CD11b antibody binding to human neutrophils. Effects of feG on basal and PAF (10^-9M) induced binding of CD11b antibody (mean ± SEM; n = 6). A. Total MFI is plotted against dose of feG. B. Net MFI (total MFI-control MFI in absence of PAF) is plotted against dose of feG. Statistically significant differences (*P < 0.05) from no peptide (0).

Figure 4

FACS analysis of CD11b antibody binding to isolated human blood neutrophils. A. Histograms of relative cell count for Isotype control (irrelevant, species-matched IgM antibody), and CD11b antibody (mouse anti-human IgG1, VIM 12 clone) for Control (no additions), PAF alone (10^-9M), feG alone (10^-11M) and feG in the presence of PAF. PAF increases fluorescence intensity relative to Control, and feG decreases the fluorescence intensity. Histograms are representative tracings. B. Dot plot of cell sample treated with 10^-9M PAF showing homogenous nature of the isolated neutrophils. The dot plot is representative of cell samples for Control and feG treated cells.

Figure 5

FACS analysis of CD16b antibody binding to isolated human blood eutrophils. A. Histograms of relative cell count for Isotype control (irrelevant, species-matched IgG antibody), and CD16b antibody (mouse anti-human IgMκ; 1D3 clone) for Control (no additions), PAF alone (10^-9M), feG alone (10^-11M) and feG in the presence of PAF. PAF only modestly increases fluorescence intensity relative to Control, and feG decreases the fluorescence intensity below those seen in Control. Histograms are representative tracings. B. Dot plot of cell sample treated with 10^-9M PAF showing homogenous nature of the isolated neutrophils. The dot plot is representative of cell samples for Control and feG treated cells.

Figure 6

feG and intracellular CD11b. Binding of CD11b antibody to membrane (external) and granular (internal) pools of neutrophils was determined in untreated controls (black columns), and in the presence of PAF alone (red columns), or PAF and feG (10^-11M) (blue columns). Results are presented as mean ± SEM (n = 6) of MFI for CD11b. Statistically significant differences (P < 0.05): # greater than control; * less than PAF.

Figure 7

feG and the binding of different antibodies to CD16b. The dose response relationship for the binding of two different antibodies against CD16b, the 1D3 and LNK16 clones, to human neutrophils. The percent change in MFI relative to PAF (10^-9M) is plotted against dose of feG. Data presented as mean ± SEM for n = 6–8. Statistically significant differences (*P < 0.05) from no peptide (0).

Figure 8

feG and binding of CD11b and CD16 antibodies to rat neutrophils. A dose response relationship for the inhibition by feG of the binding of CD11b and CD16 antibodies to PAF (10^-9M) stimulated rat neutrophils (mean ± SEM; n = 6). The percent change in MFI relative to PAF (10^-9M) is plotted against dose of feG. Statistically significant differences (*P < 0.05) from PAF in the absence of peptide.

Figure 9

Chemotaxis of rat neutrophils. Inhibition of the accumulation of neutrophils into a subcutaneously implanted carrageenan (Carr)-soaked sponge by 100 μg/kg of feG administered intraperitoneally. (mean ± SEM; n = 6–10). Statistically significant differences (P < 0.05): # greater than control; * less than Carr.

Figure 10

CD11b and CD16b antibody binding to rat neutrophils. Effect of feG on the binding of CD11b (A) and CD16 (B) antibodies to rat peritoneal (black bars) and blood (red bars) neutrophils. (mean ± SEM; n = 4–7). Four groups of ovalbumin (OA)-sensitized rats were used: unchallenged and saline treated (Cont), unchallenged and feG-treated (feG), challenged and saline-treated (OA), and challenged and feG-treated (OA+feG). Statistically significant differences (P < 0.05) where # indicates greater than OA-challenged and * indicates less than Cont, and ** less than OA.