Tissue inhibitor of metalloproteinase 1 activates normal human granulocytes, protects them from apoptosis, and blocks their transmigration during inflammation

Abstract

Urinary levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) higher than those of matrix metalloproteinase 9 (MMP-9) during acute pyelonephritis have previously been associated with a higher degree of acute inflammation and of postinfective renal scarring. The aim of the present study was to evaluate possible mechanisms by which TIMP-1 could affect the scarring process already during the acute phase of inflammation. The growth of Escherichia coli, bactericidal activity of fresh human blood, and respiratory burst, spontaneous apoptosis, and trans-basement membrane migration of normal human granulocytes were studied in vitro in the presence of different concentrations of recombinant human TIMP-1. To imitate the "normal" environment during inflammation in the kidney, granulocytes were also incubated with a conditioned medium from E. coli-stimulated renal epithelial cells. In order to compare our data with the in vivo situation, blood and urinary leukocyte levels were analyzed for 40 children with acute pyelonephritis, together with urinary MMP-9 and TIMP-1 levels. TIMP-1 at a concentration of 500 ng/ml increased the bactericidal activity of blood, increased the respiratory burst of granulocytes, decreased phosphatidylserine exposure and caspase 3 activity, which are features of spontaneous apoptosis, and inhibited granulocyte transmigration. Moreover, in the patients with pyelonephritis, MMP-9/TIMP-1 ratios in urine correlated with the degree of leukocyte transmigration. Thus, our data suggest that TIMP-1 specifically blocks the transmigration of granulocytes into urine. Entrapped and activated granulocytes, protected from apoptosis, might excessively destroy renal parenchyma and thus contribute to the pathogenesis of renal scarring following acute pyelonephritis.

FIG. 1.

Growth of E. coli in fresh human blood (Lancefield assay) (top) and in LB broth (bottom). Growth is expressed as the fold increase in bacterial number compared to the number at 0 h. At a concentration of 500 ng/ml, TIMP-1 significantly inhibited the growth of E. coli in blood (P < 0.05), indicating a stimulating effect on bactericidal activity. TIMP-1 did not significantly influence the growth of E. coli in LB broth at any of the concentrations used. Data are presented as medians and ranges, excluding outliers, from four experiments.

FIG. 2.

Respiratory burst of normal human granulocytes in PBS-glucose (PBS-Glc) and conditioned medium (Cond.med.). Median fluorescence intensity (MdFI) was used as a measure of the respiratory burst of a cell. The median respiratory burst of a control group of samples served as a reference value, and percentages of increase versus this value were calculated. TIMP-1 at a concentration of 500 ng/ml significantly increased the metabolic activity of granulocytes (P < 0.05). The addition of conditioned medium significantly increased the metabolic activity of the cells (P < 0.05). Further addition of TIMP-1 did not have a significant additive effect. Data are presented as medians and ranges, excluding outliers, from four experiments.

FIG. 3.

Morphology of normal human granulocytes stained with May-Grünwald-Giemsa stain immediately after isolation (A) and after 24 h in RPMI with 4% human serum albumin (B) and together with 500 ng of TIMP-1 per ml (C). After 24 h, typical apoptotic features, such as nuclear compaction and segregation (arrows 1), condensation of cytoplasm, nuclear fragmentation (arrows 1), and cytoplasmic blebbing (arrow 2), were seen. Defects in the cytoplasmic membrane and the presence of “cell shadows,” a consequence of cell lysis, indicated cell necrosis.

FIG. 4.

Phosphatidylserine exposure in the outer leaflet of the cytoplasmic membrane of granulocytes after 24 h in RPMI with 4% human serum albumin and in conditioned medium (Cond.med.), as measured by AV staining. Empty bars indicate the percentages of cells that were both AV and PI negative and nonapoptotic, and filled bars indicate the percentages of apoptotic, AV-positive cells, both PI positive and negative. TIMP-1 at a concentration of 500 ng/ml decreased the number of AV-positive cells (P < 0.05). In the presence of conditioned medium, the number of AV-positive cells decreased (P < 0.05). The addition of TIMP-1 further decreased the degree of phosphatidylserine exposure, with a statistically significant effect at a concentration of 500 ng/ml (P < 0.05). Data are presented as medians and ranges, excluding outliers, from four experiments.

FIG. 5.

Percentages of transmigrated granulocytes after 4 h of attraction by a conditioned medium in an in vitro transwell model. The number of transmigrated granulocytes is expressed as a percentage of the cell number added to the upper chamber at the beginning of the experiment. The transmigration of granulocytes was significantly inhibited by 20, 100, and 500 ng of TIMP-1 per ml (P < 0.05). There was no statistical difference between the effects of the different TIMP-1 concentrations used. Data are presented as medians and ranges, excluding outliers, from three experiments.

FIG. 6.

Time response of TIMP-1 effect on granulocyte transmigration. TIMP-1 (100 ng/ml) significantly inhibited the transmigration of granulocytes at both 4 and 24 h, with the largest observed difference at 4 h (P < 0.05). The negative control shows the number of transmigrated granulocytes stimulated with RPMI and human serum albumin. Data are presented as medians and ranges, excluding outliers, from three experiments.

FIG. 7.

Correlation between urinary leukocyte/blood leukocyte ratios (x axis) and MMP-9/TIMP-1 ratios (A), DMSA scores during the acute phase of inflammation (B), and DMSA scores 1 year after infection (C). We found a correlation between urinary leukocyte/blood leukocyte ratios and urinary MMP-9/TIMP-1 ratios (R = 0.40; P < 0.05). For the group of 40 children studied, there was a reverse tendency for a correlation between urinary leukocyte/blood leukocyte ratios and both acute and follow-up DMSA scores.