Neutrophil depletion blocks early collagen degradation in repairing cholestatic rat livers.

Biliary obstruction results in a well-characterized cholestatic inflammatory and fibrogenic process; however, the mechanisms and potential for liver repair remain unclear. We previously demonstrated that Kupffer cell depletion reduces polymorphonuclear cell (neutrophil) (PMN) and matrix metalloproteinase (MMP)8 levels in repairing liver. We therefore hypothesized that PMN-dependent MMP activity is essential for successful repair. Male Sprague-Dawley rats received reversible biliary obstruction for 7 days, and the rat PMN-specific antibody RP3 was administered 2 days before biliary decompression (repair) and continued daily until necropsy, when liver underwent morphometric analysis, immunohistochemistry, quantitative RT-PCR, and in situ zymography. We found that RP3 treatment did not reduce Kupffer cell or monocyte number but significantly reduced PMN number at the time of decompression and 2 days after repair. RP3 treatment also blocked resorption of type I collagen. In addition, biliary obstruction resulted in increased expression of MMP3, MMP8, and tissue inhibitor of metalloproteinase 1. Two days after biliary decompression, both MMP3 and tissue inhibitor of metalloproteinase 1 expression declined toward sham levels, whereas MMP8 expression remained elevated and was identified in bile duct epithelial cells by immunohistochemistry. PMN depletion did not alter the hepatic expression of these genes. Conversely, collagen-based in situ zymography demonstrated markedly diminished collagenase activity following PMN depletion. We conclude that PMNs are essential for collagenase activity and collagen resorption during liver repair, and speculate that PMN-derived MMP8 or PMN-mediated activation of intrinsic hepatic MMPs are responsible for successful liver repair.

Dexamethasone alters the hepatic inflammatory cellular profile without changes in matrix degradation during liver repair following biliary decompression.

BACKGROUND: Biliary atresia is characterized by extrahepatic bile duct obliteration along with persistent intrahepatic portal inflammation. Steroids are standard in the treatment of cholangitis following the Kasai portoenterostomy, and were advocated for continued suppression of the ongoing immunologic attack against intrahepatic ducts. Recent reports, however, have failed to demonstrate an improved patient outcome or difference in the need for liver transplant in postoperative patients treated with a variety of steroid regimes compared with historic controls. In the wake of progressive liver disease despite biliary decompression, steroids are hypothesized to suppress inflammation and promote bile flow without any supporting data regarding their effect on the emerging cellular and molecular mechanisms of liver repair. We have previously shown in a reversible model of cholestatic injury that repair is mediated by macrophages, neutrophils, and specific matrix metalloproteinase activity (MMP8); we questioned whether steroids would alter these intrinsic mechanisms. METHODS: Rats underwent biliary ductal suspension for 7 d, followed by decompression. Rats were treated with IV dexamethasone or saline at the time of decompression. Liver tissue obtained at the time of decompression or after 2 d of repair was processed for morphometric analysis, immunohistochemistry, and quantitative RT-PCR. RESULTS: There was a dramatic effect of dexamethasone on the inflammatory component with the initiation of repair. Immunohistochemistry revealed a reduction of both ED1+ hepatic macrophages and ED2+Kupffer cells in repair compared with saline controls. Dexamethasone treatment also reduced infiltrating neutrophils by day 2. TNF-alpha expression, increased during injury in both saline and dexamethasone groups, was markedly reduced by dexamethasone during repair (day 2) whereas IL-6, IL-10, and CINC-1 remained unchanged compared with saline controls. Dexamethasone reduced both MMP8 and TIMP1 expression by day 2, whereas MMP9, 13, and 14 were unchanged compared with sham controls. Despite substantial cellular and molecular changes during repair, collagen resorption was the same in both groups CONCLUSION: Dexamethasone has clear effects on both the hepatic macrophage populations and infiltrating neutrophils following biliary decompression. Altered MMP and TIMP gene expression might suggest that steroids have the potential to modify matrix metabolism during repair. Nevertheless, successful resorption of collagen fibrosis proceeded presumably through other MMP activating mechanisms. We conclude that steroids do not impede the rapid intrinsic repair mechanisms of matrix degradation required for successful repair.

Endotoxin alters early fetal lung morphogenesis.

BACKGROUND: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. METHOD: Gestational day 15 (E15) fetal rat lung explants (term = 22 d) were treated with either 200 ng/mL or 2 microg/mL lipopolysaccharides (LPS) with controls and examined daily by phase microscopy. After 5 d, explants were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 5 mum in the coronal plane. Immunohistochemical analysis was performed with platelet endothelial cell adhesion molecule (PECAM) to define endothelial cells, vascular endothelial growth factor (VEGF) to examine endothelial mitogenesis, and COX-2 antibodies as a marker for prostaglandin synthesis. Real-time PCR examined inducible nitric oxide synthase (iNOS), FGF9, FGF10, and FGFr2 gene expression. Air space fraction and airway epithelium were analyzed with Image J software. RESULTS: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas. By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VEGF expression was unaltered. LPS increased iNOS, but decreased FGF9, FGF10, and FGFr2 gene expression. CONCLUSIONS: These data support evidence for an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that proinflammatory stimuli may ultimately lead to abnormal pulmonary development via fibroblastic growth factor (FGF)-directed mechanisms that affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.

Disruption of interleukin-1 signaling improves the quality of wound healing.

In this study, we investigated the role of interleukin (IL)-1 signaling in wound healing. IL-1 receptor type I (IL-1R) knockout (KO) mice showed reduced fibrosis in both cutaneous and deep tissue wounds, which was accompanied by a reduction in inflammatory cellular infiltration in cutaneous but not in deep tissue wounds. There were no differences in either total collagenolytic activity or in the expression of selected matrix metalloproteinases or tissue inhibitors of metalloproteinases between the wound fluids from wild-type or IL-1R KO mice. However, wound fluids from IL-1R KO mice contained lower levels of IL-6 compared with wild-type controls. In addition, the infusion of IL-6 into wounds in IL-1R KO mice did not increase fibrosis. Skin wounds in IL-1R KO animals had lower levels of collagen and improved restoration of normal skin architecture compared with skin wounds in wild-type mice. However, neither the tensile strength of incisional skin wounds nor the rate of closure of excisional wounds differed between IL-1R KO and wild-type animals. The reduced fibrotic response in wounds from IL-1R KO mice could be reproduced by the administration of an IL-1R antagonist. These findings suggest that pharmacological interference with IL-1 signaling could have therapeutic value in the prevention of hypertrophic scarring and in the treatment of fibrotic diseases.

Hepatic macrophages promote the neutrophil-dependent resolution of fibrosis in repairing cholestatic rat livers.

BACKGROUND: Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. METHODS: Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl(3)) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. RESULTS: GdCl(3) treatment for either 24 or 48 hours before decompression reduced the numbers of ED2(+) Kupffer cells and ED1(+) inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl(3) pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl(3) treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. CONCLUSION: We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation.

Neural MMP-28 expression precedes myelination during development and peripheral nerve repair.

Mammalian matrix metalloproteinase 28 (MMP-28) is expressed in several normal adult tissues, and during cutaneous wound healing. We show that, in frog and mouse embryos, MMP-28 is expressed predominantly throughout the nervous system. Xenopus expression increases during neurulation and remains elevated through early limb development where it is expressed in nerves. In the mouse, neural expression peaks at embryonic day (E) 14 but remains detectable through E17. During frog hindlimb regeneration XMMP-28 is not initially expressed in the regenerating nerves but is detectable before myelination. Following hindlimb denervation, XMMP-28 expression is detectable along regenerating nerves before myelination. In embryonic rat neuron-glial co-cultures, MMP-28 decreases after the initiation of myelination. Incubation of embryonic brain tissue with purified MMP-28 leads to the degradation of multiple myelin proteins. These results suggest that MMP-28 plays an evolutionarily conserved role in neural development and is likely to modulate the axonal-glial extracellular microenvironment.

Kupffer cells abrogate cholestatic liver injury in mice.

BACKGROUND & AIMS: Biliary obstruction and cholestasis can cause hepatocellular apoptosis and necrosis. Ligation of the common bile duct in mice provides an excellent model in which to study the underlying mechanisms. Kupffer cells play a key role in modulating the inflammatory response observed in most animal models of liver injury. This study was performed to determine the role of Kupffer cells in the injury attending cholestasis. METHODS: Mice were not treated or were rendered Kupffer cell-depleted by intravenous inoculation of multilamellar liposome-encapsulated dichloromethylene diphosphonate, the common bile duct was ligated and divided; sham-operated animals served as controls. Similarly, interleukin-6 (IL-6)-deficient and tumor necrosis factor-receptor-deficient mice underwent bile duct ligation (BDL) or sham operations. RESULTS: Serum alanine transaminase levels were increased in all BDL mice at 3 days after surgery, but were significantly higher in IL-6-deficient mice or mice rendered Kupffer cell-depleted before ligation. Histologic examination of BDL livers showed portal inflammation, neutrophil infiltration, bile duct proliferation, and hepatocellular necrosis. Photoimage analyses confirmed more necrosis in the livers of Kupffer cell-depleted and IL-6-deficient animals. Purified Kupffer cells derived from BDL animals produced more IL-6 in culture. Similarly, Kupffer cells obtained by laser capture microdissection from the livers of BDL mice expressed increased levels of IL-6 messenger RNA. Recombinant mouse IL-6 administered 1 hour before BDL completely reversed the increased liver damage assessed otherwise in Kupffer cell-depleted mice. CONCLUSIONS: These findings indicate that Kupffer cells abrogate cholestatic liver injury by cytokine-dependent mechanisms that include the production of IL-6.

Repair after cholestatic liver injury correlates with neutrophil infiltration and matrix metalloproteinase 8 activity.

BACKGROUND: Although timely surgical treatment of liver disease can interrupt inflammation and reduce fibrosis, the mechanisms of repair are unknown. We questioned whether these mechanisms of repair include changes in the inflammatory infiltrate and associated biological activity of matrix metalloproteinases (MMPs) 8 and 2. METHODS: Rats (n >or= 3) underwent biliary ductal suspension for 7 days followed by decompression. Livers were collected after 7 days of obstruction (d0) and after 2, 5, and 7 days of repair (d2, d5, d7, respectively), and assessed morphometrically for collagen, polymorphonuclear cells (PMNs), Kupffer cells (KCs), and inflammatory mononuclear phagocytes (MNPs). In situ zymography was performed by using fluorogenic substrates for MMP-8 and MMP-2 to spatially localize enzymatic activity. RESULTS: Cholestatic injury resulted in significantly elevated (P

Expression of Xenopus XlSALL4 during limb development and regeneration.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.

Estrogen alters excitability but not morphology of a sexually dimorphic neuromuscular system in adult rats.

In rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) innervate the bulbocavernosus (BC) muscle, which surrounds the base of the penis. The SNB/BC is a sexually dimorphic, steroid-sensitive neuromuscular system, which is critically important in male reproductive behavior. Androgens are necessary for the development, morphology, and function of the SNB/BC system. However, estradiol (E) is also necessary for the development of the SNB/BC system, and E is capable of maintaining BC EMG activity in adulthood. In this study, we used electrophysiological and anatomical methods to examine estrogenic effects on BC EMG activity. We used a modified H-reflex testing method to investigate polysynaptic reflex characteristics in intact males, castrates, and castrates treated short term with estradiol benzoate (EB). Measures of EMG activity, response latency, and spike count were altered in castrates, but maintained in EB-treated castrates to the levels of intact males. Furthermore, estrogenic effects were found in EMG activity that could be isolated to the periphery of the SNB/BC system. BC NMJ size and muscle fiber area have been demonstrated to be hormone sensitive, and we examined these for possible correlates of E's effects on BC EMG activity. BC muscles of intact males, castrates, and short-term EB-treated castrates were fixed and stained with zinc iodide and osmium tetroxide. NMJ size and muscle fiber area did not differ between groups. Together, these data suggest that E treatment results in changes in the neuromuscular periphery that maintain BC EMG activity, but this effect cannot be accounted for by changes in NMJ size or muscle fiber area.

Regeneration or scarring: an immunologic perspective.

Complete regeneration of complex tissues and organs is usually precluded by fibrotic reactions that lead to scarring. Fish, salamanders, and larval anurans are among the few vertebrates capable of regenerating lost appendages, and this process seems to recapitulate ontogenic development of the structure in most respects. Recent work has revealed a capacity for excellent regeneration in certain mammalian tissues: embryonic or fetal skin and the ear of the MRL mouse. Analyses of these two systems suggest that processes of regenerative growth and patterning for the formation of new structures such as hair follicles may involve modulation of the inflammatory response to the injury in a way that reduces fibrosis and formation of scar tissue. We review evidence that this modulation includes changes in cytokine signaling and may involve properties of the extracellular matrix mediated by factors that include hyaluronic acid and "anti-adhesive substrates" such as tenascin-C. New studies and classic work on the capacity for limb regeneration in amphibians are then reviewed, focusing on the loss of this ability in prometamorphic anuran hindlimbs and the view that changing properties of the immune system may also underlie the declining regenerative potential in this system. Finally, we review recent work in comparative and developmental immunology, which raises the possibility that phylogenetic changes in regenerative capacity may be the result of evolutionary changes in cellular activities of the immune system.

Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization.

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.