Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration.

Recent studies have demonstrated that neutrophils are not a homogenous population of cells. Here, we have identified a subset of human neutrophils with a distinct profile of cell-surface receptors [CD54(high), CXC chemokine receptor 1(low) (CXCR1(low))], which represent cells that have migrated through an endothelial monolayer and then re-emerged by reverse transmigration (RT). RT neutrophils, when in contact with endothelium, were rescued from apoptosis, demonstrate functional priming, and were rheologically distinct from neutrophils that had not undergone transendothelial migration. In vivo, 1-2% of peripheral blood neutrophils in patients with systemic inflammation exhibit a RT phenotype. A smaller population existed in healthy donors ( approximately 0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54(low), CXCR1(high)) and naïve cells after activation with formyl-Met-Leu-Phe (CD54(low), CXCR1(low)). It is important that the RT phenotype (CD54(high), CXCR1(low)) is also distinct from tissue-resident neutrophils (CD54(low), CXCR1(low)). Our results demonstrate that neutrophils can migrate in a retrograde direction across endothelial cells and suggest that a population of tissue-experienced neutrophils with a distinct phenotype and function are present in the peripheral circulation in humans in vivo.

Differing mechanisms of leukocyte recruitment and sensitivity to conditioning by shear stress for endothelial cells treated with tumour necrosis factor-alpha or interleukin-1beta.

The cytokines tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1B) induce endothelial cells to recruit leukocytes. However, the exact adhesion and activation mechanisms induced by each cytokine, and their relative sensitivities to modulation by endothelial exposure to shear stress remain unclear. We cultured human umbilical vein endothelial cells (HUVEC) in glass capillaries at various shear stresses, with TNFalpha or IL-1B added for the last 4 h. Subsequently, human neutrophils were perfused over the HUVEC, and adhesion and migration were recorded. Both cytokines induced dose-dependent capture of neutrophils. However, while conditioning of HUVEC by increasing shear stress for 24 h diminished their response to TNFalpha, the response of HUVEC to IL-1B was similar at all shear stresses. The differing sensitivities were evident at levels of adhesive function and mRNA for adhesion molecules and chemokines. Analysis of nuclear factor kappaB (NF-kappaB)/Rel family of transcription factors showed that their expression and activation were modified by exposure to shear stress, but did not obviously explain differential responses to TNFalpha and IL-1B. Antibodies against selectins were effective against capture of neutrophils on TNFalpha-treated but not IL-1B-treated HUVEC. Stable adhesion was supported by beta2-integrins in each case. Activation of neutrophils occurred dominantly through CXC-chemokine receptor 2 (CXCR2) for TNFalpha-treated HUVEC, while blockade of CXCR1, CXCR2 and of platelet-activating factor receptors caused additive inhibition of migration on IL-1B-treated HUVEC. The mechanisms which underlie neutrophil recruitment, and their modulation by the haemodynamic environment, differ between cytokines. Interventions aimed against leukocyte recruitment may not operate equally in different inflammatory milieu.

Transmigrated neutrophils down-regulate the expression of VCAM-1 on endothelial cells and inhibit the adhesion of flowing lymphocytes.

As the first leukocytes recruited during inflammation, neutrophils are ideally situated to regulate the subsequent recruitment of mononuclear leukocytes. Here, we found that human neutrophils recruited by endothelial cells (EC), which had been stimulated with tumor necrosis factor alpha for 4 h, inhibited the adhesion of flowing, mixed mononuclear cells or purified lymphocytes over the subsequent 20 h but did not affect the adhesion of a secondary bolus of neutrophils. The degree of inhibition of lymphocyte adhesion increased with the duration of neutrophil-EC contact and with the number of recruited neutrophils. Antibody-blocking studies showed that lymphocyte adhesion was mediated predominantly by vascular cell adhesion molecule-1 (VCAM-1). Recruited neutrophils reduced the EC expression of VCAM-1 but not intercellular adhesion molecule-1 (ICAM-1) or E-selectin in a manner that mirrored the time- and number-dependent reduction in lymphocyte adhesion. VCAM-1 was not shed into the culture supernatant, and a panel of protease inhibitors was unable to reverse its down-regulation, indicating that it was not proteolytically degraded by neutrophils. In EC that had been in contact with neutrophils, the mRNA message for VCAM-1 but not ICAM-1 was down-regulated, indicating that alterations in transcriptional activity were responsible for the reduction in VCAM-1. Thus, under some inflammatory milieu, neutrophils may delay the recruitment of mononuclear leukocytes by regulating the expression of EC adhesion receptors.