The CXC chemokine interleukin-8 (IL-8͞CXCL8) induces rapid mobilization of hematopoietic progenitor cells (HPCs). Previously we showed that mobilization could be prevented completely in mice by pretreatment with neutralizing antibodies against the 2-integrin LFA-1 (CD11a). In addition, murine HPCs do not express LFA-1, indicating that mobilization requires a population of accessory cells. Here we show that polymorphonuclear cells (PMNs) serve as key regulators in IL-8-induced HPC mobilization. The role of PMNs was studied in mice rendered neutropenic by administration of a single injection of antineutrophil antibodies. Absolute neutropenia was observed up to 3-5 days with a rebound neutrophilia at day 7. The IL-8-induced mobilizing capacity was reduced significantly during the neutropenic phase, reappeared with recurrence of the PMNs, and was increased proportionally during the neutrophilic phase. In neutropenic mice, the IL-8-induced mobilizing capacity was restored by the infusion of purified PMNs but not by infusion of mononuclear cells. Circulating metalloproteinase gelatinase B (MMP-9) levels were detectable only in neutropenic animals treated with PMNs in combination with IL-8, showing that in vivo activated PMNs are required for the restoration of mobilization. However, IL-8-induced mobilization was not affected in MMP-9-deficient mice, indicating that MMP-9 is not indispensable for mobilization. These data demonstrate that IL-8-induced mobilization of HPCs requires the in vivo activation of circulating PMNs.metalloproteinases ͉ MMP-9 ͉ adhesion molecules ͉ bone marrow ͉ G-CSF
Minocycline is protective in models of transient middle cerebral artery occlusion (MCAO). We studied whether minocycline and doxycycline, another tetracycline derivative, provide protection in permanent MCAO. Because minocycline inhibits matrix metalloprotease-9 (MMP-9), we also compared minocycline's protective effect in wild type (wt) and MMP-9 knock-out (ko) mice. Wt FVB/N, Balb/C, and two lines of MMP-9 ko and their wt C57Bl/6 control mice were subjected to 24- or 72-hour permanent MCAO. Drug administration was started either 12 hours before or 2 hours after the onset of MCAO. Infarct size was determined by triphenyltetrazolium staining or T2-weighted MRI. Zymography was used to study the expression of MMPs. In wt strains, tetracycline treatments started before MCAO reduced the infarct size by 25% to 50%, whereas the treatment started after MCAO was not protective. Minocycline inhibited ischemia-provoked pro-MMP-9 induction in wt mice, but was not protective in MMP-9 ko mice. Pro-MMP-2 was induced by MCAO in wt and MMP-9 ko mice. MCAO-induced pro-MMP-2 was downregulated by minocycline treatment in wt mice but remained in MMP-9 ko mice at the same level as in saline-treated wt mice. Tetracyclines are protective in permanent MCAO when the treatment is started before the insult. Minocycline may provide protection by interfering with MMPs.
Cerebral malaria (CM) is a life-threatening disorder and a major medical problem in developing countries. It is caused by the sequestration of malaria-infected erythrocytes onto brain endothelia, followed by blood-brain barrier (BBB) damage and neurological deficit. In the present study, matrix metalloproteinases (MMPs) were analysed in a mouse model of CM with Plasmodium berghei ANKA. Increased numbers of gelatinase B (MMP-9)-positive cells, which were also CD11b(+), were detected in the brain. In addition, activation of gelatinase B occurred in CM brains, and not in brains of mice with non-CM. However, selective genetic knockout of gelatinase B did not alter the clinical evolution of experimental CM. To study other protease balances, the mRNA expression levels of nine matrix metalloproteinases (MMPs), five membrane-type MMPs, TNF-alpha converting enzyme (TACE) and the four tissue inhibitors of metalloproteinases (TIMPs) were analysed during CM in different organs. Significant alterations in expression were observed, including increases of the mRNAs of MMP-3, -8, -13 and -14 in the spleen, MMP-8, -12, -13 and -14 in the liver and MMP-8 and -13 in the brain. Net gelatinolytic activity, independent of gelatinase B and inhibitable with EDTA, was detected in situ in the endothelia of blood vessels in CM brains, but not in brains of mice with non-CM, suggesting that metalloproteases, different from gelatinase B, are active in the BBB environment in CM. The increase in MMP expression in the brain was significantly less pronounced after infection of C57Bl/6 mice with the noncerebral strain P. berghei NK65, but it was similar in CM-susceptible C57Bl/6 and CM-resistant Balb/C mice upon infection with P. berghei ANKA. Furthermore, in comparison with C57Bl/6 mice, a larger increase in TIMP-1 and a marked, >30-fold induction in MMP-3 were found in the brains of Balb/C mice, suggesting possible protective roles for TIMP-1 and MMP-3.
The kidney is one of the main targets of drug toxicity, and early detection of renal damage is critical in preclinical drug development. A model of cisplatin-induced nephrotoxicity in male Sprague Dawley rats treated for 1, 3, 5, 7, or 14 days at 1 mg/kg/day was used to monitor the spatial and temporal expression of various indicators of kidney toxicity during the progression of acute kidney injury (AKI). As early as 1 day after cisplatin treatment, positive kidney injury molecule-1 (Kim-1) immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin indicated the onset of proximal tubular injury in the absence of functional effects. After 3 days of treatment, Kim-1 protein levels in urine increased more than 20-fold concomitant with a positive clusterin immunostaining and an increase in urinary osteopontin. Tubular basophilia was also noted, while serum creatinine and blood urea nitrogen levels were elevated only after 5 days, together with tubular degeneration. In conclusion, tissue Kim-1 and urinary clusterin were the most sensitive biomarkers for detection of cisplatin-induced kidney damage. Thereafter, urinary Kim-1 and osteopontin, as well as clusterin immunostaining accurately correlated with the histopathological findings. When AKI is suspected in preclinical rat studies, Kim-1, clusterin, and osteopontin should be part of urinalysis and/or IHC can be performed.
To provide mechanistic insight in the induction of phospholipidosis and the appearance of the proposed biomarker di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP), rats were treated with 150 mg/kg amiodarone for 12 consecutive days and analyzed at three different time points (day 4, 9, and 12). Biochemical analysis of the serum revealed a significant increase in cholesterol and phospholipids at the three time points. Bio-analysis on the serum and urine detected a time-dependent increase in BMP, as high as 10-fold compared to vehicle-treated animals on day 12. Paralleling these increases, micro-array analysis on the liver of treated rats identified cholesterol biosynthesis and glycerophospholipid metabolism as highly modulated pathways. This modulation indicates that during phospholipidosis-induction interactions take place between the cationic amphiphilic drug and phospholipids at the level of BMP-rich internal membranes of endosomes, impeding cholesterol sorting and leading to an accumulation of internal membranes, converting into multilamellar bodies. This process shows analogy to Niemann-Pick disease type C (NPC). Whereas the NPC-induced lipid traffic jam is situated at the cholesterol sorting proteins NPC1 and NPC2, the amiodarone-induced traffic jam is thought to be located at the BMP level, demonstrating its role in the mechanism of phospholipidosis-induction and its significance for use as a biomarker.
The detection of drug-induced hepatotoxicity remains an important safety issue in drug development. A liver-specific microRNA species, microRNA-122 (miR-122), has recently shown potential for predicting liver injury in addition to the standard hepatic injury biomarkers. The objective of this study was to measure miR-122 together with several other liver markers in distinct settings of acute liver toxicity in rats to determine the value of miR-122 as a biomarker for liver injury in this species. Rats were exposed to 3 well-established liver toxicants (acetaminophen, allyl alcohol, and a-naphthyl isothiocyanate), a liver-enzyme inducer (phenobarbital), or a cardiotoxicant (doxorubicin). There was a clear increase in plasma miR-122 following administration of acetaminophen, allyl alcohol, and a-naphthyl isothiocyanate. The response of miR-122 paralleled that of other markers and was consistent with liver injury as indicated by histopathological evaluation. Furthermore, the changes in miR-122 were detected earlier than standard liver injury markers and exhibited a wide dynamic range. In contrast, miR-122 responses to phenobarbital and doxorubicin were low. Based on these findings, miR-122 shows significant promise and may provide added value for assessing liver toxicity in drug development.
Gelatinase B or matrix metalloproteinase‐9 (MMP‐9) is stored in the tertiary granules of polymorphonuclear leukocytes. These cells are key effectors in acute inflammatory diseases such as sepsis. Endotoxin leads to rapid release of gelatinase B from these granules in vitro and in vivo, but the role of this enzyme in bacterial sepsis and endotoxin shock remains unclear. We studied the clinical course of endotoxinemia and its relation with the expression of gelatinase B from the pool of circulating leukocytes in adult as well as in young mice in a model of endotoxin‐induced shock and compared wild‐type with gelatinase B‐deficient mice. The gelatinase B‐deficient mice were resistant to endotoxin shock, which implies that specific MMP‐9 inhibition constitutes anapproach for the treatment of septic shock syndromes.
Leukocytosis is a physiopathological mechanism primarily to combat infections, whereas stem cell mobilization is induced for therapeutical purposes. Both processes are dependent on the balance between leukocyte and stem cell retention and mobilization. The retention is mediated by the specific architecture of the bone marrow, adhesion molecules and the production of chemokines in the bone marrow, which attract escaped immature cells to the marrow. Mobilization is the effect of the action of "peripheral" chemokines, such as interleukin-8 (IL-8 or CXCL8) and the remodeling of the matrix and basement membranes by matrix enzymes, such as gelatinase B (MMP-9). Recent studies lead to the conclusion that neutrophils, IL-8/CXCL8 and gelatinase B/MMP-9 play control roles in leukocytosis and stem cell mobilization. Neutrophils are the predominant circulating leukocyte type and IL-8/CXCL8 is the major neutrophil chemoattractant in humans. Gelatinase B and no gelatinase A is rapidly released from prestored granules after activation of neutrophils by IL-8/CXCL8. Moreover, neutrophils do not produce TIMP-1 and can chemically activate latent progelatinase B. Activated gelatinase B catalyses the aminoterminal truncation of IL-8/CXCL8 into a tenfold more potent chemokine. This implies that, when IL-8/CXCL8 appears in the circulation, the bone marrow is instructed to release neutrophils and concomitantly stem cells. These studies suggest that IL-8/CXCL8 and gelatinase B/MMP-9 are targets for the modulation of stem cell mobilization.
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