Effect of a kinin B2 receptor antagonist on LPS- and cytokine-induced neutrophil migration in rats

Abstract

1 This study examines the involvement of kinins in neutrophil migration into rat subcutaneous air pouches triggered by lipopolysaccharide (LPS), as well as the putative roles played by kinin B(1) and B(2) receptors, tumour necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and selectins in this response. 2 LPS (5 ng to 10 micro g cavity(-1)) injected into the 6-day-old pouch induced a dose- and time-dependent neutrophil migration which peaked between 4 and 6 h, and was maximal following the dose of 100 ng cavity(-1) (saline: 0.46+/-0.1; LPS: 43+/-3.70 x 10(6) cells cavity(-1) at 6 h). 3 Bradykinin (BK) (600 nmol) injected into the pouch of saline-treated rats induced only modest neutrophil migration (0.73+/-0.16 x 10(6) cells cavity(-1)). A more robust response to BK (3.2+/-0.25 x 10(6) cells cavity(-1)) was seen in animals pretreated with captopril, but this was still smaller than the responses to IL-1beta or TNF-alpha (15 pmol: 23+/-2.2 x 10(6) and 75 pmol: 29.5+/-2 x 10(6) cells cavity(-1), respectively). Nevertheless, the B(1) agonist des-Arg(9)-BK (600 nmol) failed to induce neutrophil migration. 4 HOE-140 (1 and 2 mg kg(-1)), a B(2) receptor antagonist, reduced LPS-induced neutrophil migration. HOE-140 also reduced the neutrophil migration induced by BK, but had no effect on the migration promoted by IL-1beta or TNF-alpha. des-Arg(9)-[Leu(8)]-BK, B(1) receptor antagonist was ineffective in changing neutrophil migration caused by any of these stimuli. 5 Neutrophil migration induced by LPS or BK was reduced by interleukin-1 receptor antagonist (IL-1ra) (1 mg kg(-1)), sheep anti-rat TNF serum (anti-TNF serum) (0.3 ml cavity(-1)), and the nonspecific selectin inhibitor fucoidin (10 mg kg(-1)). 6 TNF-alpha levels in the pouch fluid were increased by LPS or BK injection, peaking at 0.5-1 h and gradually declining thereafter up to 6 h. IL-1beta levels increased steadily throughout the 6 h period. HOE-140 markedly inhibited the rise in IL-1beta and TNF-alpha levels in pouch fluid triggered by both stimuli. 7 These results indicate that BK participates importantly in selectin-dependent neutrophil migration into the air pouch triggered by LPS in the rat, by stimulating B(2) receptors coupled to synthesis/release of TNF-alpha and IL-1beta.

Figure 1

Time-course of neutrophil migration induced by LPS, 100 ng cavity⁻¹, injected in a volume of 1 ml into the rat air pouch. Control animals received the same volume of sterile saline (only value for 6 h is shown in figure). The bars represent the mean±s.e. mean number of neutrophils (6–9 animals) present in pouch fluid collected at the times indicated. ^*Significantly different from control animals, P<0.05, Scheffé's test.

Figure 2

Effect of DALBK and HOE-140 on neutrophil migration induced by injection of LPS (left panel) or BK (right panel) into the rat air pouch. DALBK and HOE-140 were injected subcutaneously (s.c.), at doses indicated, 30 min before LPS (100 ng cavity⁻¹) or BK (600 nmol cavity⁻¹). Control animals received s.c. injection of sterile saline (SAL). Captopril (5 mg kg⁻¹, s.c.) was given 1 h before BK as stimulus in the pouch. The bars represent the mean±s.e. mean number of neutrophils (6–9 animals) detected in pouch fluid collected 6 h after LPS or BK. ^*Significantly different from saline-treated animals (SAL), P<0.05, Scheffé's test.

Figure 3

Effect of DALBK or HOE-140 on neutrophil migration induced by IL-1β or TNF-α into the rat air pouch. DALBK or HOE-140, each at 1 mg kg⁻¹, was injected subcutaneously (s.c.) 30 min before IL-1β or TNF-α. The two control groups received either sterile saline alone into the pouch (C) or were pretreated s.c. with sterile saline instead of B₁ or B₂ receptor antagonist. The bars represent the means±s.e. mean number of neutrophils (6–9 animals) detected in pouch fluid collected 6 h after IL-1β or TNF-α. All values of cytokine-treated groups differed from C, P<0.05, Scheffé's test.

Figure 4

Effect of IL-1 receptor antagonist (IL-1ra), sheep anti-rat TNF serum (anti-TNF) or fucoidin on neutrophil migration induced by LPS (left panel) or BK (right panel) into the rat air pouch. IL-1ra (1 mg kg⁻¹) or anti-TNF (0.3 ml cavity⁻¹) was coinjected with the stimuli into the air pouches. An additional and similar dose of IL-1ra was also given 1 h later in that particular group. Fucoidin (10 mg kg⁻¹, i.v.) was injected 15 min before the stimuli. Control animals received sterile saline instead of IL-1ra, anti-TNF or fucoidin. Animals that received BK as a stimulus were pretreated with captopril (5 mg kg⁻¹, s.c., 1 h beforehand). Bars represent the mean±s.e. mean number of neutrophils (6–9 animals) detected in pouch fluid collected 6 h after LPS or BK. ^*Significantly different from saline treated animals, P<0.05, Scheffé's test.

Figure 5

Effect of HOE-140 on the levels of IL-1β and TNF-α in the fluid from rat air pouch injected with either LPS or BK. HOE-140 (1 mg kg⁻¹) was injected subcutaneously (s.c.) 30 min before LPS (100 ng cavity⁻¹, panels a and c) or BK (600 nmol cavity⁻¹, panels b and d). The appropriate control groups received sterile saline either s.c. (instead of HOE-140) or into the pouch (SAL; instead of LPS or BK), and the fluid was collected 6 h later. Animals that received BK as the stimulus were pretreated with captopril (5 mg kg⁻¹, s.c., 1 h beforehand). The bars represent the means±s.e. mean of duplicates for each animal (n=4). ^*Significantly different from saline treated animals, P<0.05, Student's t-test.