ABSTRACT
An increase in circulating neutrophils is a characteristic feature of many inflammatory reactions and is a result of the rapid mobilization of neutrophils from the bone marrow, driven by inflammatory mediators, including the ELR + CXC chemokines. In this paper, using a combination of light and electron microscopy and an in situ perfusion system of the rat femoral bone marrow, we examined this mobilization process in detail. We show that mobilization of neutrophils stimulated by the CXC chemokine, rat MIP-2, involves neutrophil migration from the haematopoietic compartment of the bone marrow across the bone marrow sinusoidal endothelium via a transcellular route. The critical role of the bone marrow sinusoidal endothelium in regulating neutrophil mobilization was demonstrated by artificially disrupting the bone marrow endothelial barrier by treatment with cytochalasin D, which results in the non-selective release of leucocytes from the bone marrow. In contrast, inhibiting the activity of p38 mitogen-activated protein kinase, inhibited both MIP-2 stimulated chemotaxis of bone marrow neutrophils in vitro and neutrophil mobilization in situ while, a broad spectrum matrix metalloproteinase inhibitor, BB94, had no effect on neutrophil mobilization. These results support the hypothesis that neutrophil migration drives their mobilization and highlights the function of the sinusoidal endothelium in regulating this process.
Subject(s)
Bone Marrow/physiology , Chemokine CXCL2/pharmacology , Chemokines, CXC/pharmacology , Neutrophils/physiology , Animals , Cell Movement , Cytochalasin D/pharmacology , Endothelium , Lymphocyte Activation , Male , Matrix Metalloproteinase Inhibitors , Nucleic Acid Synthesis Inhibitors/pharmacology , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Rats , Rats, Sprague-Dawley , Thiophenes/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The acute release of neutrophils from the bone marrow is a critical step in their trafficking to sites of inflammation. This process is stimulated by systemically acting inflammatory mediators, such as the CXC chemokines. In this study we have used a novel in situ perfusion system of the rat femoral bone marrow to directly investigate the role of specific adhesion molecules in chemokine-stimulated neutrophil mobilization. We show here that neutrophils mobilized in response to rat macrophage inflammatory protein-2 (MIP-2) shed L-selectin and expressed significantly higher levels of CD11b and CD49d. However, inhibition of L-selectin sheddase activity with KD-IX-73-4 had no effect on the number of neutrophils mobilized in response to rat MIP-2. Blockade of CD18, using a neutralizing monoclonal antibody (mAb), did not inhibit neutrophil mobilization but unexpectedly increased the rate and number of neutrophils released from the bone marrow in response to chemokine, suggesting that CD18 could play a role in neutrophil retention within the bone marrow. Blockade of CD49d using either a selective mAb or a specific antagonist resulted in a dramatic inhibition (> 75%) of the chemokine-stimulated neutrophil mobilization from the bone marrow. These data reveal contrasting roles for CD18 and CD49d in the retention and release of neutrophils from the bone marrow.
Subject(s)
Bone Marrow Cells/metabolism , Bone Marrow/metabolism , Cell Movement/drug effects , Chemokines, CXC/administration & dosage , Integrin alpha4/metabolism , Neutrophils/metabolism , Animals , Antibodies, Monoclonal/administration & dosage , Bone Marrow Cells/cytology , CD11b Antigen/biosynthesis , CD18 Antigens/metabolism , Chemokine CXCL2 , Chemokines, CXC/metabolism , Dipeptides/administration & dosage , Enzyme Inhibitors/administration & dosage , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Hydroxamic Acids/administration & dosage , Inflammation/metabolism , L-Selectin/metabolism , Male , Neutrophils/cytology , Rats , Rats, Sprague-DawleyABSTRACT
In this study we provide evidence that the SDF-1alpha/CXCR4 chemokine axis is involved in both the retention of neutrophils within the bone marrow and the homing of senescent neutrophils back to the bone marrow. We show that the functional responses of freshly isolated human and murine neutrophils to CXCR2 chemokines are significantly attenuated by SDF-1alpha, acting via CXCR4. As a consequence, the mobilization of neutrophils from the bone marrow in vivo by the CXCR2-chemokine, KC, was dramatically enhanced by blocking the effects of endogenous SDF-1alpha using a specific CXCR4 antagonist. As neutrophils age, they upregulate expression of CXCR4 and acquire the ability to migrate toward SDF-1alpha. We show here that these senescent CXCR4(high) neutrophils preferentially home to the bone marrow in vivo in a CXCR4-dependent manner, suggesting a previously undefined mechanism for the clearance of senescent neutrophils from the circulation.
