Regulation of matrix metalloproteinases and their inhibitor genes in lipopolysaccharide-induced endotoxemia in mice

Abstract

An imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia.

Figure 1.

Time course of MMP and TIMP gene expression in the liver after LPS injection. Mice were given a single i.p. injection of LPS (20 μg or 1 mg in 100 μl of PBS). Poly(A)⁺ RNA was isolated from the liver at the times shown and 1 μg used to determine MMP or TIMP mRNA levels by RPA as described in Materials and Methods. A: Time course of gene expression after injection of 20 μg of LPS. Autoradiograms showing the RPA-labeled probe sets used in this analysis are shown at the sides. The RPL 32 probe does not appear in the part of the RPA shown here. Note that the hybridized liver target mRNAs that lack the cloning site of the probe migrate at a lower molecular size. Two animals were used for each time point. The autoradiographic exposure times were 16 hours (MMP) and 6 hours (TIMP). B: Quantitative analysis of MMP and TIMP gene expression shown in A (20 μg LPS, fine curve) and after 1 mg of LPS (bold line, three mice were used at each time point, X MMP and TIMP gene expression in three mice that died at 15 to 18 hours after LPS). Charts are given for RNAs that are either induced or up-regulated more than twofold. For constitutively expressed RNAs that are up-regulated after LPS injection, here MT1-MMP and TIMP-3, the y axis label shows the fold up-regulation. For induced genes, the y axis label indicates arbitrary units.

Figure 2.

Quantitative analysis of MMP and TIMP gene expression the organs of mice after injection of different doses of LPS (20 μg LPS, fine curve) and after 1 mg of LPS (bold line, three mice were used at each time point, X MMP and TIMP gene expression in three mice that died at 15 to 18 hours after LPS). A: MMP and TIMP gene expression in the spleen after different doses of LPS. B: Quantitative analysis of MMP and TIMP gene expression in the kidney after different doses of LPS. C: Quantitative analysis of MMP and TIMP gene expression in the brain after different doses of LPS. The RPAs and quantitative analysis was performed as described in Materials and Methods and the legend to Figure 1 ▶ .

Figure 3.

Comparison of MMP gene expression in LPS-resistant C3H/HeJ and LPS-sensitive C3H/FeJ mice. Mice were given an i.p. injection of LPS (20 μg) and were killed 8 hours later. RPA was performed using 1 μg poly(A)⁺ RNA from each organ as described in Materials and Methods.

Figure 4.

Comparison of TIMP gene expression in LPS-resistant C3H/HeJ and LPS-sensitive C3H/FeJ mice. Mice were given an i.p. injection of LPS (20 μg) and were killed 8 hours later. RPA was performed using 1 μg poly(A)⁺ RNA from each organ as described in Materials and Methods.

Figure 5.

Cellular localization of collagenase 3 RNA in various organs of control and LPS-treated mice. In situ hybridization was performed as described in Materials and Methods. Liver: control (A), LPS-injected (B and C). The probe hybridizes to cells resembling von Kupffer-cells (arrowheads). Original magnification: ×20 (A and B), ×60 (C). Spleen: control (D); LPS-injected (E). Original magnification ×20. Kidney: control (F); hemorrhage after LPS injection (G; arrowheads). H&E; original magnification, ×10. Control (H); LPS-injected (I). Original magnification, ×10.

Figure 6.

Cellular localization of TIMP-2 RNA in various organs of control and LPS-treated mice. In situ hybridization was performed as described in Materials and Methods. Spleen: control (A); LPS-injected (B). Original magnification, ×40. Kidney: control (C); glomerulum, LPS-injected, note the labeled cells around a glomerulum (D, arrows). Original magnification, ×40. Brain: control (E); expression of TIMP-1 RNA in the meninges after LPS-injection (F; arrows). Original magnification, ×10.

Figure 7.

MMP activity in different organs of mice after LPS-injection. Mice were killed 12 hours after i.p. LPS injection (20 μg) and organs were frozen in liquid nitrogen. Frozen organs were homogenized in lysis buffer and protein content determined as described in Materials and Methods. Unreduced samples were fractionated on 10% acrylamide gels containing 1 mg/ml gelatin. Subsequently, gelatin zymography was performed as described in Materials and Methods. Protein loading was 50 μg for brain, 20 μg for kidney, 6 μg for spleen, and 7 μg for liver. The constitutive gelatinolytic activity in the brain is not easily visible in this reproduction.

Figure 8.

Localization of gelatinolytic activity in different organs of mice after LPS-injection. Mice were killed 16 hours after i.p. LPS-injection (100 μg) and organs were frozen in liquid nitrogen. Cryostat sections were over-laid with a solution containing quenched fluorescent gelatin and sections were examined 16 to 20 hours later. Gelatinolytic activity becomes apparent as fluorescence. Liver: control (A); LPS-injected (B). Original magnification, ×40. Spleen: control (C); follicle (f), the border between the follicle and red pulp is demarcated by the dotted white line; LPS-injected (D). Note the fluorescent plaques in the follicle (f) after LPS injection (arrows). Original magnification, ×40. Kidney: control (E), note the autofluorescence of the vessel walls; LPS-injected (F). Original magnification, ×20. Brain (dentate gyrus): control (G); control, addition of 10 mmol/L EDTA to the assay solution abolished the neuronal fluorescence (H). Original magnification, ×20.