Chemokine regulation of neutrophil function in surgical inflammation.

BACKGROUND: Morbidity and even mortality correlate closely with major injury that causes a systemic inflammatory response. Cytokines and bioactive molecules present at the inflammatory site induce this response and regulate neutrophil proinflammatory responses. The CXC chemokines, important for neutrophil recruitment and activation, include interleukin 8 (IL-8), granulocyte chemotactic protein 2 (GCP-2), and epithelial cell-derived neutrophil attractant 78 (ENA-78). They induce neutrophil responses via 2 cell-surface receptors, CXCR-1 and CXCR-2. All 3 chemokines bind CXCR-2 with high affinity. Only IL-8 and GCP-2 bind CXCR-1 with high affinity. HYPOTHESIS: The CXC chemokines regulate neutrophil responses differently. METHODS: Pretreatment of neutrophils from healthy volunteers with IL-8, GCP-2, or ENA-78; measured IL-8-induced migration; and tumor necrosis factor alpha (TNF-alpha)-induced peroxide production. RESULTS: Flow cytometry and radioligand binding data indicate that IL-8, GCP-2, and ENA-78 equivalently reduced CXCR-1 and CXCR-2 cell surface expression by 34% to 54%. All treatments decreased affinity of both receptors 1.5- to 2-fold. However, only IL-8 pretreatment inhibited chemotaxis to 10-nmol/L IL-8 (mean +/- SE inhibition, 62%+/-6%). Although IL-8 and GCP-2, but not ENA-78, suppressed TNF-alpha-induced oxidant production (mean +/- SE inhibition, 42%+/-8% and 40%+/-23%, respectively), only GCP-2 inhibited the oxidative response to complement fragment C5a, and to the bacterial cell wall peptide N-formyl-methionyl-leucyl-phenylalanine. CONCLUSIONS: The CXC chemokines regulate neutrophil proinflammatory functions differently. A thorough understanding of mechanisms for modulating neutrophil responses in inflammation will aid the development of interventions that reduce morbidity and mortality associated with severe trauma and sepsis.

Tumor necrosis factor alpha regulates CXC chemokine receptor expression and function.

The alpha chemokine family is central to the participation of neutrophils in the acute inflammatory response. These substances interact with neutrophils through two cell surface receptors, CXCR-1 and CXCR-2 (formally known as IL-8R-1 and IL-8R-2). We investigated the possible regulatory effects of tumor necrosis factor alpha (TNFalpha) pretreatment on CXCR-1 and CXCR-2. To this end, we examined these receptors with flow cytometry, radioligand binding, Northern blot analyzes, calcium mobilization, and chemotaxis experiments on human neutrophils. In flow cytometry experiments, TNFalpha pretreatment substantially decreased cell surface CXCR-2 receptor levels but showed partial recovery at 120 min. On the other hand, CXCR-1 receptor levels had a sharp decline at 15 min and maintained that level to 120 min. Northern blot analyzes showed that mRNA levels of both IL-8 receptors were essentially unchanged after 45 min of TNFalpha pretreatment, but declined markedly following 2 h of pretreatment. Chemotaxis experiments on cells treated with TNFalpha for 5-120 min showed a substantial down-regulation of chemotaxis to IL-8 and GROalpha. This was noted to be much greater than the decline in cell surface receptors. Calcium mobilization experiments revealed minimal inhibition of the IL-8-induced increase in calcium after pretreatment with TNFalpha, but the response to NAP-2 was substantially inhibited. The data demonstrate differential regulation of the IL-8 receptor.

Mononuclear cell line THP-1 internalizes bactericidal/permeability-increasing protein by a non-receptor-mediated mechanism consistent with pinocytosis.

BACKGROUND: Bactericidal/permeability-increasing protein (BPI) binds lipopolysaccharide and neutralizes its toxic effects in vitro and in endotoxemic animals. Our recent work identified physiologically significant interactions between BPI, lipopolysaccharide, and mononuclear cells. OBJECTIVE: To determine whether the interaction between BPI and mononuclear cells is receptor mediated. DESIGN: Labeled BPI was incubated with THP-1 cells in the presence of up to 100-fold excess of unlabeled BPI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were performed to evaluate competitive binding and total uptake of BPI. Crosslinking was performed to determine whether BPI binds to a single protein entity. Acid washing experiments and flow cytometric analysis were performed to determine whether BPI remains on the cellular surface. Finally, flow cytometry analysis was used to determine whether BPI incubation with THP-1 cells affects the surface expression of the lipopolysaccharide-binding protein-lipopolysaccharide receptor CD14. RESULTS: Labeled BPI uptake was not inhibited by the presence of 100-fold excess of unlabeled BPI at 37 degrees C or 4 degrees C in the presence of azide. Uptake was not saturable under either condition with incubation concentrations up to 10 microgram/mL. Cross-linking did not show BPI bound to a single entity. Acid washing and flow cytometry experiments disclosed rapid internalization of BPI. Finally, BPI uptake by THP-1 cells had no effect on the surface expression of CD14. CONCLUSIONS: Bactericidal/permeability-increasing protein is rapidly internalized by mononuclear cells in a nonspecific fashion not saturable at very high doses, which is consistent with pinocytosis. This process may represent a disposal mechanism for lipopolysaccharide in closed-space infections and may be partially responsible for the rapid clearance of BPI from the peripheral circulation.

Regulation of TNF-alpha receptor expression on human neutrophils by various cell activating factors.

Since tumor necrosis factor-alpha (TNF-alpha) has been implicated in a wide range of inflammatory states, we sought to examine the regulation of TNF-alpha receptors on human neutrophils (PMNs). We were particularly interested in the relationship of TNF-alpha binding characteristics to various known neutrophil-priming stimuli such as temperature, N-formylmethionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), C5ades Arg, and human recombinant C5a (HrC5a). Competition binding studies indicated that there was no significant change in receptor number or affinity due to the temperature used for neutrophil isolation. PMNs isolated at 4 or 25 degrees C had a single class of receptors with a Kd of 83.1 +/- 3.3 vs 90.0 +/- 8.3 pmole/liter and a similar number of receptors, 1575 +/- 99 vs 1876 +/- 279 receptors/cell. However, when either group of isolated PMNs was further incubated in buffer at 37 degrees C for 15 min, a significant increase in Kd (4-37 degrees C, 147.9 +/- 23.7 pmole/liter; 25-37 degrees C, 165.2 +/- 29.2 pmol/liter) and a decrease in receptor number (4-37 degrees C, 1116 +/- 86 receptors/cell; 25-37 degrees C, 957 +/- 71 receptors/cell) were seen. To determine the effect of other activating substances such as fMLP, PMA, C5ades Arg, HrC5a, 4 degrees C isolated PMNs were used.(ABSTRACT TRUNCATED AT 250 WORDS)