Characterization of the priming effect by pituitary canine growth hormone on canine polymorphonuclear neutrophil granulocyte function

Abstract

In this report, we demonstrate that canine growth hormone (cGH) is capable of priming canine polymorphonuclear neutrophil granulocytes (PMN) in a manner resembling that of human PMN. The cGH influences important functions that are involved in the process of recruitment of PMN, i.e., shape change, chemotaxis, CD11b/CD18 expression, adhesion, and subsequent transendothelial migration. Also, intracellular O(2)(-) production was evaluated. We investigated the priming effect by incubating PMN with purified pituitary cGH at various concentrations (10 to 800 microg/liter). The capacity for shape change was significantly (P < 0.05) enhanced, whereas the chemotactic response under agarose was significantly (P < 0.05) reduced. The chemotactic migration in Boyden chambers (10-microm-thick polycarbonate filter; lower surface count technique) was significantly (P < 0.05) enhanced, presumably due to cGH-induced hyperadhesiveness to the lower surface of the filters. The adhesion in albumin-coated microtiter plates and adherence to canine pulmonary fibroblasts were significantly (P < 0.05) increased, and the increased adhesion resulted in a significant (P < 0.01) increase in transendothelial migration using canine jugular vein endothelial cells. The increase in adhesion was associated with a significant increase in CD11b/CD18 expression. Furthermore, intracellular O(2)(-) production was significantly enhanced in response to both phorbol myristate acetate (P < 0.01) and opsonized zymosan (P < 0.05). In the absence of a PMN-stimulating agent, cGH did not influence the effector functions investigated except for an increased expression of CD11b/CD18.

FIG. 1

Enhancement of shape change (mean values ± standard errors of means) of canine PMN incubated with cGH stimulated with 10 nM IL-8 (⧫) compared to 0 μg of cGH per liter (significance: ∗, P < 0.05; ∗∗, P < 0.01; paired t test; n = 10). Unstimulated shape change (●) was not influenced by cGH (P > 0.05 [n = 10] compared to 0 μg of cGH per liter).

FIG. 2

Decreased chemotactic migration under agarose of canine PMN (box-whiskers plot) incubated with cGH for 30 min at 37°C toward 100 nM IL-8 compared to control (significance: ∗, P < 0.05; ∗∗, P < 0.01; Wilcoxon signed rank test; n = 10).

FIG. 3

cGH (400 μg/liter) enhances the chemotactic migration of canine PMN toward IL-8 (10 nM) in Boyden chambers as determined by the LSC technique (∗, P < 0.05; paired t test; n = 10) (mean values ± standard errors of means).

FIG. 4

cGH (≥100 μg/liter) enhanced PMA (100 nM)-stimulated adhesion of canine PMN in albumin-coated microtiter plates (mean values ± standard errors of means) compared to control (left panel) (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; paired t test; n = 10). cGH did not influence the unstimulated adhesion (right panel) (P > 0.05; paired t test; n = 10).

FIG. 5

cGH (400 μg/liter) enhanced PAF (200 nM)-stimulated adherence of canine PMN (mean values ± standard errors of means) to canine pulmonary fibroblasts compared to control (∗∗, P < 0.01; paired t test; n = 10). Unstimulated adhesion was not influenced by cGH (P > 0.05; paired t test; n = 10).

FIG. 6

Influence of cGH (400 μg/liter) on the interaction of canine PMN with canine jugular vein endothelial cells. (A) No influence by cGH on the adhesion of PMN to unstimulated endothelial cells compared to control (P > 0.05; paired t test; n = 10). (B) cGH enhances the interaction (adherence and transmigration) between PMN and LPS-stimulated endothelial cells compared to control (∗, P < 0.05; paired t test; n = 10). (C) cGH enhanced PMN transmigration across LPS-stimulated endothelial cells compared to control (∗∗, P < 0.01; paired t test; n = 10) when percent PMN was based on the number of PMN in initial contact with the endothelium. (D) No influence of cGH on the PMN transmigration across LPS-stimulated endothelium when percent PMN was based on the number of interacting PMN (P > 0.05; paired t test; n = 10) (mean values ± standard errors of means).

FIG. 7

cGH (400 μg/liter) enhances the O₂⁻ production of canine PMN when stimulated with PMA (100 nM) or OPZ (∼10⁷ particles/ml) as determined by the NBT reduction assay (∗, P < 0.05; ∗∗, P < 0.01; paired t test; n = 10). The unstimulated O₂⁻ production is not influenced by cGH (P > 0.05; paired t test; n = 10) (mean values ± standard errors of means). O.D., optical density.