Metalloproteinases Are Involved in Lipopolysaccharide– and Tumor Necrosis Factor-–Mediated Regulation of CXCR1 and CXCR2 Chemokine Receptor Expression by Masud H. Khandaker, Gordon Mitchell, Luoling Xu, Joseph D. Andrews, Rajkumari Singh, Harry Leung, Joaquı́n Madrenas, Stephen S.G. Ferguson, Ross D. Feldman, and David J. Kelvin Blood Volume 93(7):2173-2185 April 1, 1999 ©1999 by American Society of Hematology

Distribution of CXCR1 expression on IL-8–, LPS-, and TNF-–treated neutrophils. Distribution of CXCR1 expression on IL-8–, LPS-, and TNF-–treated neutrophils. Purified peripheral blood PMNs were incubated for 1 hour at 37°C in media alone (RPMI/10% fetal calf serum) or stimulated with IL-8 (500 ng/mL), LPS (100 ng/mL), or TNF- (50 ng/mL). Cells were then stained with FITC-conjugated Ab to CXCR1 and examined by confocal microscopy using an oil immersion lens at 600× magnification. (A) Cellular distribution of maximal CXCR1 fluorescence for each treatment is shown at left, and transmission light microscopy of the same cell is shown at right, with size bars representing 10 μm. (B) Mean membrane v cytoplasm CXCR1 staining intensity is plotted for n = 15 (±SEM) cells per treatment group. *Statistical significance (P < .05) using 1-way ANOVA for membrane luminosity of control untreated groupv treated groups. **Statistically significant increase (P < .05; one-way ANOVA) in cytoplasm luminosity vmembrane luminosity of IL-8–treated cells. Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Dose-response of proteinase inhibitors on CXCR1 and CXCR2 induced downmodulation. Dose-response of proteinase inhibitors on CXCR1 and CXCR2 induced downmodulation. Purified peripheral blood PMNs were preincubated with various concentrations of (A) 1,10-phenanthroline (B) EDTA, or (C) bestatin for 30 minutes followed by the addition of LPS (100 ng/mL) (•), TNF- (50 ng/mL) (▪), or IL-8 (500 ng/mL) (▴) for 1 hour at 37°C. The x-axis indicates inhibitor concentration (μg/mL). The y-axis indicates percent inhibition of downmodulation. Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

1,10-Phenanthroline blocks LPS- and TNF-–stimulated release of CXCR1 cleavage products. 1,10-Phenanthroline blocks LPS- and TNF-–stimulated release of CXCR1 cleavage products. Purified peripheral blood PMNs were preincubated with 1,10-phenanthroline (0.5 mmol/L) for 30 minutes in media (RPMI/10% fetal calf serum) followed by the addition of LPS (100 ng/mL) or TNF- (50 ng/mL) for 1 hour at 37°C. Cell supernatants were isolated, and the proteins were assayed on a 10% SDS-polyacryamide gel and electrophoretically transferred to PVDF membranes. The PVDF membrane shown was immunoblotted with polyclonal Ab recognizing the carboxy-terminal amino acids 341-359 of the CXCR1 molecule. Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

Effect of proteinase inhibitors on IL-8–directed neutrophil chemotaxis. Effect of proteinase inhibitors on IL-8–directed neutrophil chemotaxis. Purified peripheral blood PMNs were untreated (▪) or preincubated with 1,10-phenanthroline (0.5 mmol/L, ▨), EDTA (5 mmol/L, ░), or bestatin (100 μmol/L, □) for 30 minutes at 37°C followed by the addition of LPS (100 ng/mL), TNF- (50 ng/mL), or IL-8 (500 ng/mL) for 1 hour at 37°C. The migration assay was then performed. The data represent a single experiment from 4 performed. Results are the mean ± SEM migrated cells counted from three high-powered fields (400×) obtained in three replicates. *Statistical significance (P < .05) using one-way ANOVA for control untreated v treated groups. Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology

LPS-, TNF-–, and IL-8–induced CXCR downregulation is not due to cell death. LPS-, TNF-–, and IL-8–induced CXCR downregulation is not due to cell death. Purified peripheral blood PMNs were incubated for 3 hours at 37°C in media alone (RPMI/10% fetal calf serum) or stimulated with LPS (100 ng/mL), TNF- (50 ng/mL), IL-8 (500 ng/mL), or dexamethasone (100 mmol/L). Propidium iodide staining and CXCR1 staining was measured using two-color parameter flow cytometry. Data are represented as contour plots with the x-axis indicating CXCR1 fluorescence intensity measured on a log10 scale and the y-axis indicating propidium iodide fluorescence intensity measured on a log10 scale. MFI values for CXCR1 staining are indicated in the bottom right panel of each contour plot. MFI values for propidium iodide staining are indicated in the top right panel of each contour plot. Similar data were observed for CXCR2 expression. Masud H. Khandaker et al. Blood 1999;93:2173-2185 ©1999 by American Society of Hematology