BRIEF COMMUNICATION

Reduced Leukocyte Adhesion Response and Absence of Slow Leukocyte Rolling in Interleukin-8 Receptor-Deficient Mice☆

Chemokines have long been implicated in the initiation and amplification of inflammatory responses by virtue of their role in leukocyte chemotaxis. The expression of one of the receptors for these chemokines, CXCR2, on a variety of cell types and tissues suggests that these receptors may have a broad functional role under both constitutive conditions and in the pathophysiology of a number of acute and chronic diseases. With the development of several pharmacological, immunological and genetic tools to study CXCR2 function, an important role for this CXC chemokine receptor subtype has been identified in chronic obstructive pulmonary disease (COPD), asthma and fibrotic pulmonary disorders. Interference with CXCR2 receptor function has demonstrated different effects in the lungs including inhibition of pulmonary damage induced by neutrophils (PMNs), antigen or irritant-induced goblet cell hyperplasia and angiogenesis/collagen deposition caused by lung injury. Many of these features are common to inflammatory and fibrotic disorders of the lung. Clinical trials evaluating small molecule CXCR2 antagonists in COPD, asthma and cystic fibrosis are currently underway. These studies hold considerable promise for identifying novel and efficacious treatments of pulmonary disorders.

The murine chemokine receptor 2 (mCXCR2) controls resistance to urinary tract infection. We have previously shown that mCXCR2 knockout mice develop severe acute pyelonephritis and renal tissue damage with sub-epithelial neutrophil entrapment. In this study we examined the relative importance of neutrophil- and epithelial-specific mCXCR2 expression for bacterial clearance in bone marrow chimeric mice infected with uropathogenic Escherichia coli. Mice expressing mCXCR2 on their neutrophils responded rapidly to experimental urinary tract infection, clearing the infection from the kidneys. Mice lacking epithelial mCXCR2, however, showed delayed exit of neutrophils from the tissues. Mice lacking neutrophil mCXCR2 and mice with no mCXCR2 had no neutrophil recruitment and bacterial clearance. Mice expressing mCXCR2 only in their epithelial cells had a transient epithelial chemokine response; however, neutrophil recruitment was inhibited and bacteria grew without constraint. Our study shows that the expression of mCXCR2 on hematopoietic cells was crucial for bacterial clearance, while its expression on non-bone marrow-derived cells influenced the neutrophil response. These results emphasize the importance of mCXCR2 for the innate defense against urinary tract infection.

This chapter is concerned with the microcirculation in inflammation, which is modulated and influenced by platelet–leukocyte interactions (see above) and by the coagulation system. Thrombin, the main product of the coagulation cascade, activates vascular endothelial cells, mononuclear leukocytes and platelets through the protease-activated receptors. All tissues with a microcirculation can participate in an inflammatory response. Aulus Celsus introduced four of the five cardinal symptoms of inflammation, rubor, tumor, calor, dolor, or redness, swelling, heat, and pain. All four prominently involve the microcirculation: redness and heat reflect vasodilation; dolor is caused by the stimulation of nociceptors in the inflamed tissue, which can be amplified by swelling caused by microvascular leakage. Microcirculation consists of arterioles, capillaries, and venules. Arterioles have a smooth muscle cell-containing wall and, with few exceptions, show a divergent branching pattern, meaning that blood flows from one arteriole into two branches at each bifurcation. Postcapillary venules have no smooth muscle and are instead lined by pericytes. Venules collect blood from capillaries, which start from a divergent bifurcation and end at a confluent junction. The most important function of the microcirculation during the inflammatory response is to ensure rapid and abundant delivery of leukocytes to the inflamed tissue site. Depending on the type of inflammatory stimulus, these include neutrophils, monocytes, lymphocytes, eosinophils, and basophils. Endothelial cells control the localization of the inflammatory response as well as leukocyte and plasma protein access to tissues. At sites of inflammation, endothelial cells upregulate many inflammatory adhesion molecules, cytokines, and chemokines.

Chemokines, including CXCL1, participate in neutrophil recruitment by triggering the activation of integrins, which leads to arrest from rolling. The downstream signaling pathways which lead to integrin activation and neutophil arrest following G-protein–coupled receptor engagement are incompletely understood. To test whether Gα_i2 is involved, mouse neutrophils in their native whole blood were investigated in mouse cremaster postcapillary venules and in flow chambers coated with P-selectin, ICAM-1, and CXCL1. Gnai2^−/− neutrophils showed significantly reduced CXCL1-induced arrest in vitro and in vivo. Similar results were obtained with leukotriene B₄ (LTB₄). Lethally irradiated mice reconstituted with Gnai2^−/− bone marrow showed a similar defect in chemoattractant-induced arrest as that of Gnai2^−/− mice. In thioglycollate-induced peritonitis and lipopolysaccaride (LPS)–induced lung inflammation, chimeric mice lacking Gα_i2 in hematopoietic cells showed about 50% reduced neutrophil recruitment similar to that seen in Gnai2^−/− mice. These data show that neutrophil Gα_i2 is necessary for chemokine-induced arrest, which is relevant for neutrophil recruitment to sites of acute inflammation.

Inflammation plays a pivotal role in atherosclerosis. In addition to being a risk marker for cardiovascular disease, much recent data support a role for C-reactive protein (CRP) in atherogenesis. Interleukin-8 (IL-8), a member of the CXC chemokines promotes monocyte–endothelial cell adhesion and arrest and is abundant in atherosclerotic plaques. However, there is a paucity of data examining the effect of CRP on IL-8 secretion in human aortic endothelial cells (HAEC). In this report, we show that incubation of HAEC with CRP resulted in a time and dose-dependent increase in IL-8 protein and mRNA via transcription. In contrast to human umbilical vein endothelial cells, monocyte-chemoattractant protein-1 expression in HAEC was not affected by CRP. Furthermore, CRP upregulated NF-kappa B activity in HAEC and inhibitors of NF-kappa B significantly reversed the upregulation of IL-8 by CRP. Blocking antibodies to IL-8 significantly decreased monocyte–endothelial cell adhesion induced by CRP (31%, P < 0.01). In conclusion, this study makes the novel observation that CRP induces IL-8 synthesis and secretion in HAEC via upregulation of NF-kappa B activity.

Previously it was shown that β₂-integrins are necessary for slow leukocyte rolling in inflamed venules. In this study, mice that are deficient for either one of the β₂-integrins, α_Lβ₂ (LFA-1) or α_Mβ₂ (Mac-1), were used to determine which of the β₂-integrins are responsible for slowing rolling leukocytes. The cremaster muscles of these mice were treated with tumor necrosis factor-α and prepared for intravital microscopy. The average rolling velocities in venules were elevated in LFA-1^−/−mice (11.0 ± 0.7 µm/s) and Mac-1^−/− mice (10.1 ± 1.1 µm/s) compared to wild-type mice (4.8 ± 0.3 µm/s;P < .05), but were lower than in CD18^−/−mice (28.5 ± 2.1 µm/s). When both LFA-1 and Mac-1 were absent or blocked, rolling velocity became dependent on shear rate and approached that of CD18^−/− mice. In addition, leukocyte adhesion efficiency was decreased in LFA-1^−/− mice to near CD18^−/− levels, but decreased only slightly in Mac-1^−/− mice. Thus, both LFA-1 and Mac-1 contribute to slowing down rolling leukocytes, although LFA-1 is more important than Mac-1 in efficiently inducing firm adhesion.