Association between kinin B(1) receptor expression and leukocyte trafficking across mouse mesenteric postcapillary venules

Abstract

Using intravital microscopy, we examined the role played by B(1) receptors in leukocyte trafficking across mouse mesenteric postcapillary venules in vivo. B(1) receptor blockade attenuated interleukin (IL)-1beta-induced (5 ng intraperitoneally, 2 h) leukocyte-endothelial cell interactions and leukocyte emigration ( approximately 50% reduction). The B(1) receptor agonist des-Arg(9)bradykinin (DABK), although inactive in saline- or IL-8-treated mice, caused marked neutrophil rolling, adhesion, and emigration 24 h after challenge with IL-1beta (when the cellular response to IL-1beta had subsided). Reverse transcriptase polymerase chain reaction and Western blot revealed a temporal association between the DABK-induced response and upregulation of mesenteric B(1) receptor mRNA and de novo protein expression after IL-1beta treatment. DABK-induced leukocyte trafficking was antagonized by the B(1) receptor antagonist des-arg(10)HOE 140 but not by the B(2) receptor antagonist HOE 140. Similarly, DABK effects were maintained in B(2) receptor knockout mice. The DABK-induced responses involved the release of neuropeptides from C fibers, as capsaicin treatment inhibited the responses. Treatment with the neurokinin (NK)(1) and NK(3) receptor antagonists attenuated the responses, whereas NK(2), calcitonin gene-related peptide, or platelet-activating factor receptor antagonists had no effect. Substance P caused leukocyte recruitment that, similar to DABK, was inhibited by NK(1) and NK(3) receptor blockade. Mast cell depletion using compound 48/80 reduced DABK-induced leukocyte trafficking, and DABK treatment was shown histologically to induce mast cell degranulation. DABK-induced trafficking was inhibited by histamine H(1) receptor blockade. Our findings provide clear evidence that B(1) receptors play an important role in the mediation of leukocyte-endothelial cell interactions in postcapillary venules, leading to leukocyte recruitment during an inflammatory response. This involves activation of C fibers and mast cells, release of substance P and histamine, and stimulation of NK(1), NK(3), and H(1) receptors.

Figure 1

Videomicroscopy images of leukocyte trafficking responses in mouse mesenteric postcapillary venules in vivo in response to (A) saline (0.25 ml intraperitoneally, 2 h), (B) IL-1β (5 ng intraperitoneally, 2 h), and (C) IL-1β (5 ng intraperitoneally, 24 h).

Figure 2

Leukocyte–endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in response to B₁ receptor activation with DABK (30 nmol intraperitoneally). (A) Leukocyte rolling velocity, (B) leukocyte adhesion, and (C) leukocyte emigration were examined 2 and 4 h after DABK or saline administration. Mice received either DABK (•) or saline (▪) 24 h after IL-1β (time = 0). The effect of DABK in saline- (○, 24 h) or IL-8–treated (□, 24 h) mice is also shown. Data are mean ± SEM for n = 5–8 animals per group. *P < 0.05 versus saline-treated values.

Figure 2

Leukocyte–endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in response to B₁ receptor activation with DABK (30 nmol intraperitoneally). (A) Leukocyte rolling velocity, (B) leukocyte adhesion, and (C) leukocyte emigration were examined 2 and 4 h after DABK or saline administration. Mice received either DABK (•) or saline (▪) 24 h after IL-1β (time = 0). The effect of DABK in saline- (○, 24 h) or IL-8–treated (□, 24 h) mice is also shown. Data are mean ± SEM for n = 5–8 animals per group. *P < 0.05 versus saline-treated values.

Figure 2

Leukocyte–endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in response to B₁ receptor activation with DABK (30 nmol intraperitoneally). (A) Leukocyte rolling velocity, (B) leukocyte adhesion, and (C) leukocyte emigration were examined 2 and 4 h after DABK or saline administration. Mice received either DABK (•) or saline (▪) 24 h after IL-1β (time = 0). The effect of DABK in saline- (○, 24 h) or IL-8–treated (□, 24 h) mice is also shown. Data are mean ± SEM for n = 5–8 animals per group. *P < 0.05 versus saline-treated values.

Figure 3

Identification of leukocytes by (A and B) hematoxylin and eosin staining and (C and D) mast cells by toluidine blue staining of mesenteries from mice treated with either (A, B, and D) DABK (2 h) 24 h after IL-β or (C) saline (2 h). (A) Mesenteric section showing a postcapillary venule (v) and arteriole (a); original magnification: ×20. (B) Postcapillary venule (v) with a predominance of polymorphonuclear cells (arrowheads) either attached within or emigrated from the venule (original magnification: ×100). (C) Nondegranulated mast cells (arrowheads) in saline-treated mice (original magnification: ×40; inset original magnification: ×100). (D) Degranulated mast cells (arrowheads) in DABK/IL-1–treated mice (original magnification: ×40; inset original magnification: ×100).

Figure 3

Identification of leukocytes by (A and B) hematoxylin and eosin staining and (C and D) mast cells by toluidine blue staining of mesenteries from mice treated with either (A, B, and D) DABK (2 h) 24 h after IL-β or (C) saline (2 h). (A) Mesenteric section showing a postcapillary venule (v) and arteriole (a); original magnification: ×20. (B) Postcapillary venule (v) with a predominance of polymorphonuclear cells (arrowheads) either attached within or emigrated from the venule (original magnification: ×100). (C) Nondegranulated mast cells (arrowheads) in saline-treated mice (original magnification: ×40; inset original magnification: ×100). (D) Degranulated mast cells (arrowheads) in DABK/IL-1–treated mice (original magnification: ×40; inset original magnification: ×100).

Figure 3

Identification of leukocytes by (A and B) hematoxylin and eosin staining and (C and D) mast cells by toluidine blue staining of mesenteries from mice treated with either (A, B, and D) DABK (2 h) 24 h after IL-β or (C) saline (2 h). (A) Mesenteric section showing a postcapillary venule (v) and arteriole (a); original magnification: ×20. (B) Postcapillary venule (v) with a predominance of polymorphonuclear cells (arrowheads) either attached within or emigrated from the venule (original magnification: ×100). (C) Nondegranulated mast cells (arrowheads) in saline-treated mice (original magnification: ×40; inset original magnification: ×100). (D) Degranulated mast cells (arrowheads) in DABK/IL-1–treated mice (original magnification: ×40; inset original magnification: ×100).

Figure 3

Identification of leukocytes by (A and B) hematoxylin and eosin staining and (C and D) mast cells by toluidine blue staining of mesenteries from mice treated with either (A, B, and D) DABK (2 h) 24 h after IL-β or (C) saline (2 h). (A) Mesenteric section showing a postcapillary venule (v) and arteriole (a); original magnification: ×20. (B) Postcapillary venule (v) with a predominance of polymorphonuclear cells (arrowheads) either attached within or emigrated from the venule (original magnification: ×100). (C) Nondegranulated mast cells (arrowheads) in saline-treated mice (original magnification: ×40; inset original magnification: ×100). (D) Degranulated mast cells (arrowheads) in DABK/IL-1–treated mice (original magnification: ×40; inset original magnification: ×100).

Figure 4

DABK causes dose-related changes in (A) leukocyte rolling velocity, (B) adhesion, and (C) emigration in mouse mesenteric postcapillary venules. Mice received DABK (10–30 nmol in 0.25 ml saline, intraperitoneally) 24 h after IL-1β (•; 5 ng in 0.25 ml saline intraperitoneally) or saline alone (○; 0.25 ml intraperitoneally). Leukocyte responses were examined 2 h after DABK treatment. Data are mean ± SEM for n = 5–8 animals per group. *P < 0.05 versus saline-treated values.

Figure 5

Upregulation of the B₁ receptor by IL-1β in mouse mesenteric vascular bed. (A) Detection of B₁ receptor mRNA by RT-PCR. B₁ receptor (435 bp) and GAPDH (363 bp) PCR products amplified from polyA⁺ RNA extracted from mesenteric vascular beds from either saline- (0.25 ml, intraperitoneally) or IL-1β–treated (5 ng in 0.25 ml saline, intraperitoneally) mice. Positive PCR control is a mouse B₁ receptor cDNA plasmid. Results are representative of tissue from at least three animals per group. Inset: PCR product yield as a function of initial amount of cDNA. Serial dilutions of cDNA before PCR results in a log plot in which a linear relationship between starting amount of cDNA and product intensity is observed. B₁ receptor and GAPDH curves are parallel (P > 0.05). (B) Western blot analysis of the expression of mouse B₁ receptor using the IgG purified from A15C (reference 27; dilution 1:20,000) on mesenteric tissues (20 μg protein per lane). Mesenteric tissue were taken from either saline- (0.25 ml intraperitoneally) or IL-1β–treated (5 ng in 0.25 ml saline intraperitoneally) mice. Results are representative of tissue from four experiments. + anti B₁ represents inhibition of detection of the 39-kD band when the antibody is preincubated (overnight at 4°C) with the A15C peptide at a concentration of 10 μg/ml. Molecular mass marker proteins were run simultaneously. Their molecular masses (kD) are shown on the left side of the figure.

Figure 5

Upregulation of the B₁ receptor by IL-1β in mouse mesenteric vascular bed. (A) Detection of B₁ receptor mRNA by RT-PCR. B₁ receptor (435 bp) and GAPDH (363 bp) PCR products amplified from polyA⁺ RNA extracted from mesenteric vascular beds from either saline- (0.25 ml, intraperitoneally) or IL-1β–treated (5 ng in 0.25 ml saline, intraperitoneally) mice. Positive PCR control is a mouse B₁ receptor cDNA plasmid. Results are representative of tissue from at least three animals per group. Inset: PCR product yield as a function of initial amount of cDNA. Serial dilutions of cDNA before PCR results in a log plot in which a linear relationship between starting amount of cDNA and product intensity is observed. B₁ receptor and GAPDH curves are parallel (P > 0.05). (B) Western blot analysis of the expression of mouse B₁ receptor using the IgG purified from A15C (reference 27; dilution 1:20,000) on mesenteric tissues (20 μg protein per lane). Mesenteric tissue were taken from either saline- (0.25 ml intraperitoneally) or IL-1β–treated (5 ng in 0.25 ml saline intraperitoneally) mice. Results are representative of tissue from four experiments. + anti B₁ represents inhibition of detection of the 39-kD band when the antibody is preincubated (overnight at 4°C) with the A15C peptide at a concentration of 10 μg/ml. Molecular mass marker proteins were run simultaneously. Their molecular masses (kD) are shown on the left side of the figure.

Figure 6

The effect of B₁ or B₂ receptor blockade on the changes in leukocyte kinetics induced by DABK (30 nmol). Mice were injected with IL-1β (5 ng) 24 h before DABK together with saline, des-arg¹⁰HOE 140 (DAHOE 140; B₁ receptor antagonist, 25 nmol) or HOE 140 (B₂ receptor antagonist, 25 nmol). Leukocyte kinetics (A) rolling velocity, (B) adhesion, and (C) emigration were examined 2 h after DABK treatment. Data are mean ± SEM for n = 5–8 animals per group. *P < 0.05 versus saline-treated values.

Figure 8

Effect of a range of selective receptor antagonists on substance P– (7.5 nmol intraperitoneally), compound 48/80– (1.2 mg/kg intraperitoneally), and PAF-induced (1 μg intraperitoneally) changes in (A) leukocyte rolling velocity, (B) leukocyte adhesion, and (C) leukocyte emigration at the 2-h time point. Values from saline-treated control animals are V _WBC = 58.7 ± 9.2 μm/s; adherent leukocytes = 1.0 ± 0.5cells/100 μm; emigrated leukocytes = 0.7 ± 0.1 cells/100 × 50 μm²). The following specific treatments were used: SR 140333 (0.1 μmol/kg intravenously, 30 min); MEN 11420 (0.1 μmol/kg intravenously, 15 min); SR 142801 (4 μmol/kg intravenously, 15 min); triprolidine (1 mg/kg intraperitoneally, 20 min); and WEB 2086 (5 mg/kg intravenously, 20 min). Data are mean ± SEM for n = 6 animals per group. *P < 0.05 versus substance P–, compound 48/80–, or PAF-treated values.

Figure 7

Effect of capsaicin, compound 48/80, and a range of selective receptor antagonists on DABK-induced (30 nmol intraperitoneally) changes in (A) leukocyte rolling velocity, (B) leukocyte adhesion, and (C) leukocyte emigration at the 2-h time point. Values from saline-treated control animals were V _WBC = 52.8 ± 6.6 μm/s; adherent leukocytes = 1.9 ± 0.5 cells/100 μm; emigrated leukocytes = 0.2 ± 0.1 cells/100 × 50 μm². The following specific treatments were used: capsaicin (50 mg/kg subcutaneously, 4 d); CGRP-(8–37) (300 nmol/kg intravenously, 15 min); SR 140333 (0.1 μmol/kg intravenously, 30 min); MEN 11420 (0.1 μmol/kg intravenously, 15 min); SR 142801 (4 μmol/kg intravenously, 15 min); WEB 2086 (5 mg/kg intravenously, 20 min); compound 48/80 (1.2 mg/kg intraperitoneally, 72 h [reference 23]); and triprolidine (1 mg/kg intraperitoneally, 20 min [reference 26]). Data are mean ± SEM for n = 6 animals per group. *P < 0.05 versus DABK-treated values.

Figure 9

Leukocyte kinetics. (A) rolling velocity, (B) adhesion, and (C) emigration in the mesenteric vascular bed of BK2r^−/− (white bars) and BK2r^+/+ (black bars) mice. Mice were injected with IL-1β (5 ng intraperitoneally) 24 h before DABK (30 nmol intraperitoneally). No significant difference (P > 0.05) in any of the parameters measured was observed in BK2r^−/− compared with BK2r^+/+ mice 2 h after DABK treatment. Data are mean ± SEM for n = 5–8 animals per group. *P < 0.05 versus saline-treated values within the same genetic background.