Neurokinin-1 Receptor Knockout Mice Microvasculature: An In Vivo Study Using Inflamed, But Not Normal, Cutaneous in Mediating Neutrophil Accumulation in the Neurokinin-1 Receptor Agonists Are Involved

We have used tachykinin neurokinin-1 receptor (NK 1 receptor) knockout mice to learn of the link between NK 1 receptors and neutrophil accumulation in normal naive skin, as compared with inﬂamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK 1 knockout mice, as expected. However, in contrast to IL-1 b (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1 b -induced neutrophil accumulation was similar in wild-type and knockout mice, but a signiﬁcant ( p < 0.05) contributory effect of added NK 1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK 1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inﬂamed skin, under certain circumstances, the NK 1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inﬂammatory process. We investigated responses to two inﬂammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was signiﬁcantly attenuated ( p < 0.001) in carrageenin- but not zymosan-induced inﬂammation in NK 1 knockout mice. The carrageenin (500 m g)-induced response was inhibited ( p < 0.05) by a NK 1 receptor antagonist, SR140333 (480 nmol/kg i.v. at 2 5 min), in the wild-type group. The bradykinin B 1 and B 2 receptor antagonists (desArg 9 [Leu 8 ]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals ( p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK 1 receptor-dependent neutrophil accumulation in inﬂamed mouse skin. The Journal of Immunology, 2000, 164: 5424–5429. The homogenate was centrifuged at 13,000 3 g for 5 min (Mi- crocentaur MSE, Scotlab, Strathclyde, Lanarkshire, U.K.). Supernatants (1 ml) were then frozen at 2 18°C. A previously validated method for mea- surement of neutrophils in the rat skin (4, 9, 10) was adapted for mouse skin. Neutrophil accumulation was measured by comparing myeloperoxi- dase activity in extracts from known amounts of mouse neutrophils and compared with that at skin sites. Myeloperoxidase activity was determined using the H 2 O 2 oxidation of 3,3 9 ,5,5 9 -tetramethylbenzidine (Bionostics, Skybio, Wyboston, Beds, U.K.). Reactions were performed in a 96-well microtiter plate at room temperature. OD readings at 620 nm were taken at 5-min intervals for 30 min with a microplate reader (Anthos HTIII, Labtech International, Saltzburg, Austria). These values were plotted, and a reaction rate (OD/time) was measured from the initial slope of the curve. A cali- bration curve was then produced, with the rate of reaction plotted against the number of neutrophils in the standard samples. This was used to con- vert reaction rates to number of neutrophils for the skin sample homogenates. l (car-rageenin) NK 1 re- ceptor