Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils

Abstract

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 +/- 11.6% of counted cells) and type III (24.7 +/- 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-8) M) and leukotriene B4 (LTB4, 10(-8) M). L-NAME (but not D-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and LTB4 (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0. 05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by L-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.

Figure 1

Immunolocalization of type II (A–C) and type III (D–G) NOS in cytoplasmic granules of purified rat peritoneal eosinophils. NOS was detected by using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (blue reaction product) and counterstained with eosin. Ring-like nuclear shape (A, C, D, and F) predominated over bilobular nuclei (A, B, D, E, and G) for both NOS isotypes. Nomarski micrographs. [Original magnifications: ×33 (A and D); ×333, (B, C, E–G).]

Figure 2

The inhibition by N^ω-nitro-l-arginine methyl ester (l-NAME; cross-hatched columns) of in vitro rat eosinophil migration induced by fMLP (5 × 10⁻⁸ M) compared with either the control value (solid column) or the inactive enantiomer d-NAME (open columns). l-NAME (0.1–1.0 mM) or d-NAME (0.1–1.0 mM) was incubated with the rat eosinophil suspension for 30 min at 37°C. Each experiment was carried out in triplicate. Eosinophil migration is expressed as the mean number of migrated cells per HPF. The results are shown as the mean ± SEM (n = 3). ∗, P < 0.05 compared with either the control value or d-NAME.

Figure 3

The inhibition by TRIM (open columns) and AMT (crosshatched columns) of in vitro rat eosinophil migration induced by fMLP (5 × 10⁻⁸ M). The control value (migration in absence of both TRIM and AMT) is shown by the solid bar. TRIM (0.1–1.0 mM) or AMT (0.1–1.0 mM) was incubated with the rat eosinophil suspension for 30 min at 37°C. Each experiment was carried out in triplicate. Eosinophil migration is expressed as the mean number of migrated cells per HPF. The results are shown as the mean ± SEM (n = 3). ∗, P < 0.05 compared with the control value.

Figure 4

l-NAME, AMT, and TRIM significantly inhibit the in vitro rat eosinophil migration induced by LTB₄ (10⁻⁸ M) compared with control values (MEM; solid bar). Each of these compounds was used at concentrations of 0.1 (open bars) and 1.0 (crosshatched bars) mM. Each experiment was carried out in triplicate. Eosinophil migration is expressed as the mean number of migrated cells per HPF. The results are shown as the mean ± SEM (n = 3). ∗, P < 0.05 compared with the control value.