In vivo responses of macrophages and perisinusoidal cells to cholestatic liver injury.

We investigated the response of macrophages and perisinusoidal (Ito) cells (PSCs) during the development of secondary biliary cirrhosis after ligation and division of the common bile duct. Liver tissue was obtained from three groups of male Wistar rats: 1) untreated controls (n = 3); 2) common bile duct-ligated (CBDL) animals (n = 15); and 3) sham-operated controls (n = 15). Material from animal groups 2 and 3 was obtained on days 3, 7, 14, 21, and 28 after operation; in all animals 5-bromo-2-deoxyuridine was administered intraperitoneally before death. Monocytes and macrophages were detected using the monoclonal antibody ED1 and tissue macrophages using the antibody ED2. Cell proliferation within the macrophage population was demonstrated by double labeling for ED2 and incorporated 5-bromo-2-deoxyuridine. PSCs were demonstrated in tissue sections by immunolocalization of desmin; proliferating PSCs were identified by double labeling for desmin and incorporated 5-bromo-2-deoxyuridine. Evidence of phenotypic modulation of PSCs was sought using anti-alpha-smooth muscle actin (alpha-SMA) antibody. Increased numbers of ED1- and ED2-positive cells were seen in CBDL animals at all time points. Local proliferation of macrophages could be identified and reached a peak at day 3, thereafter falling toward control values. Compared with those of controls, livers of CBDL animals showed increased numbers of desmin-positive PSCs in periportal zones from day 3 on, reaching a peak at day 14 (127.8 +/- 10.99 cells/0.635 mm2) and followed by a plateau. PSC proliferation peaked at days 3 and 7 (labeling indices 11.2% and 11.2%, respectively) and thereafter fell toward control values; no expansion of the PSC population was seen in sham-operated rats. Increased alpha-SMA-positive cells were also noted from day 3, with a peak at day 21 (231.1 +/- 11.52 cells/0.635 mm2) and followed by a plateau. En face labeling experiments in days 14, 21, and 28 CBDL animals showed cells co-expressing alpha-SMA and desmin and cells expressing alpha-SMA alone. These results indicate that in response to chronic cholestatic liver injury, PSCs proliferate and undergo phenotypic modulation toward "myofibroblast-like" cells. The kinetics of the response are similar to those of the ED2-positive cell population in keeping with a hypothesis that PSC proliferation and activation may be mediated by factors released by macrophages in response to various forms of liver injury. We conclude that the responses of macrophages and PSCs to cholestatic injury are similar to those after toxin-induced hepatocyte necrosis.     

Hepatic macrophages in liver homeostasis and diseases-diversity,plasticity and therapeutic opportunities

Macrophages, which are key cellular components of the liver, have emerged as essential players in the maintenance of hepatic homeostasis and in injury and repair processes in acute and chronic liver diseases. Upon liver injury, resident Kupffer cells (KCs) sense disturbances in homeostasis, interact with hepatic cell populations and release chemokines to recruit circulating leukocytes, including monocytes, which subsequently differentiate into monocyte-derived macrophages (MoMϕs) in the liver. Both KCs and MoMϕs contribute to both the progression and resolution of tissue inflammation and injury in various liver diseases. The diversity of hepatic macrophage subsets and their plasticity explain their different functional responses in distinct liver diseases. In this review, we highlight novel findings regarding the origins and functions of hepatic macrophages and discuss the potential of targeting macrophages as a therapeutic strategy for liver disease.     

Macrophage Populations in Different Stages of Induced Hepatic Metastases in Rats: an Immunohistochemical Analysis

Macrophages play a role in the host defence against cancer. Little is known about changes in macrophage populations during early metastatic growth. To evaluate the distribution, number and phenotype of macrophages in the development of hepatic metastases in a rat model (Wag/Rij rats and syngeneic CC531 colon carcinoma cell line), an immunohistochemical study was performed with the monoclonal antibodies ED1 (monocytes, and all macrophages), ED2 (resident tissue macrophages, like Kupffer cells) and ED3 (a subpopulation of macrophages which may play a role in the recruitment of lymphocytes). OX19 and Hisl4 were used to identify lymphocytes. In this study a new monoclonal antibody CC52 is described, which recognizes the CC531 tumour cell line. Liver metastases were induced by injection of CC53I colon carcinoma cells into a mesenteric vein. Rats were killed at various intervals. Results show three major macrophage populations during hepatic tumour growth: (1) on day 3, infiltrates are observed around the micrometastases, which contain mainly newly recruited macrophages (ED1⁺ and ED2⁻); (2) after 7 days, ED3-positive (ED3 ⁺) macrophages together with T lymphocytes are found in the infiltrates; (3) an increase in the number of ED2-positive (ED2⁺) Kupffer cells is observed in the liver parenchyma after 14 days. In conclusion, the present results suggest that various populations of macrophages, newly recruited (ED1⁺) as well as resident Kupffer cells (ED2⁺), are involved in the immune response against tumour cell deposits in the liver.     

Local macrophage proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis

Glomerular crescent formation is a feature of aggressive forms of glomerulonephritis. The conventional view of crescent formation within Bowman's space involves proliferation of parietal epithelial cells and the recruitment of blood monocytes. However, the potential role of local macrophage proliferation in this process has not been investigated. The current study examines macrophage proliferation within Bowman's space on the basis of expression of the proliferating cell nuclear antigen (PCNA) in a rat model of crescentic glomerulonephritis (accelerated anti-GBM disease). ED1⁺ macrophages accounted for 42% of cells within early cellular crescents, and 38% of these crescent macrophages were proliferating on the basis of PCNA expression. Macrophages became the dominant cell population in advanced cellular and fibrocellular crescents (64–71%), and there was a significant increase in the level of macrophage proliferation, with 62% and 67% of ED1⁺ macrophages expressing the PCNA, respectively. This high level of macrophage proliferation was confirmed by incorporation of bromo-deoxyuridine and the presence of mitotic figures within crescents. Indeed, macrophages accounted for 73% of all proliferating cells within advanced and fibrocellular crescents. Macrophage proliferation within Bowman's space was a local event, as shown by a lack of proliferating monocytes in the circulation, the presence of mitotic figures within crescents and a reciprocal relationship between the numbers of ED1⁺PCNA⁺ cells within Bowman's space compared with that in the capillary tuft during the progression from early to advanced and fibrocellular crescents. In conclusion, this study has changed the conventional view of the pathogenesis of crescent formation in glomerulonephritis with the demonstration of substantial local macrophage proliferation within Bowman's space. It is proposed that local proliferation is a major mechanism of macrophage accumulation within crescents and plays an important role in the progression of epithelial-dominated early cellular crescents to macrophage-dominated advanced and fibrocellular cellular crescents.     

Phenotypic modulation of perisinusoidal cells following acute liver injury: a quantitative analysis.

Expression of the alpha-(smooth muscle) isoform of actin (alpha-SMA) by non-parenchymal cells in rat liver was studied following induction of acute liver injury using a single sublethal dose of carbon tetrachloride (CCl4). In normal liver, alpha-SMA immunoreactivity was identified in the smooth muscle cells of hepatic arteries and in the walls of portal and hepatic vein branches. Occasional alpha-SMA-containing stellate shaped cells were found in acinar zone 3 but most perisinusoidal cells (PSCs) did not express this protein. In CCl4-treated animals, there was an increase in the number of immunoreactive cells in perivenular zones, reaching a peak at day 3 following exposure to the toxin. These cells were morphologically identical to desmin-positive PSCs and the kinetics of the responses of alpha-SMA-positive and desmin-positive cells were similar. In en face labelling experiments, evidence of co-expression of alpha-SMA and desmin by non-parenchymal cells was obtained, although some desmin-positive PSCs did not appear to express alpha-SMA. These results suggest that PSCs rapidly undergo phenotypic modulation in response to acute liver injury with acquisition of alpha-SMA expression. It is proposed that these phenotypic changes coincide with functional alterations, such activated 'myofibroblast-like' cells being responsible for the enhanced matrix protein synthesis necessary for tissue repair.     

Proliferation of macrophage subpopulations in the adult rat: comparison of various lymphoid organs

Adult male Lewis rats received a single intravenous injection of 5-bromo-2'-deoxyuridine (BRDU) to label all proliferating cells in the S-phase of the cell cycle. Various lymphoid organs were removed 1 and 24 hr after injection to assess local proliferation and migration of newly formed cells, respectively. In cell suspensions, surface staining was performed for macrophage subsets (ED1, ED2, ED3), and the DNA label BRDU was detected by a monoclonal antibody. Local proliferation of ED1+ macrophages occurred in all organs investigated with the exception of the blood. Bone marrow outweighed the other organs by far; in addition to the proliferating ED1+ promonocytes, the bone marrow also contained BRDU-labeled ED2+ macrophages. Newly formed ED1+ monocytes migrated into lymphoid organs such as the mesenteric lymph nodes and spleen where they comprised about 90% of newly formed macrophages. In the spleen, ED3+ macrophages seemed to be renewed by local proliferation, whereas in the mesenteric lymph nodes these cells were replaced by immigration. The heterogeneity of macrophages was further demonstrated by the different renewal of splenic macrophages. ED1+ and ED3+ cells were replaced in a matter of days, whereas it would probably take several months to renew ED2+ cells.     

Local macrophage proliferation in multinucleated giant cell and granuloma formation in experimental Goodpasture's syndrome.

Granuloma is a specialized form of inflammatory reaction featuring focal macrophage and T-cell accumulation and multinucleated giant cell formation. It is widely held that macrophage accumulation within granulomatous lesions results from recruitment of blood monocytes, whereas proliferation of monocyte/macrophages makes little contribution to this process. The present study of macrophage proliferation within immunologically induced granulomas in rat experimental Goodpasture's syndrome challenges the conventional view. In this disease, granulomatous lesions in the kidney and lung contained 60 to 70% macrophages of an ED1+ED2-ED3-blood monocyte phenotype. However, double immunohistochemistry showed that up to 75% of ED1+ macrophages within granulomatous lesions were proliferating on the basis of proliferating cell nuclear antigen expression and bromodeoxyuridine incorporation. In contrast, no proliferating cell nuclear antigen expression or bromodeoxyuridine incorporations was detected in blood monocytes, indicating that proliferation of ED1+ED2-ED3- cells was a localized event within granulomatous lesions. A second finding of note was that almost all ( > 95%) nuclei within multinucleated giant cells were positive for proliferating cell nuclear antigen, but these nuclei lacked bromodeoxyuridine incorporation. This suggests a novel mechanism of multinucleated giant cell formation involving fusion of macrophages in G1 phase, which then halts progression into S phase of the cell cycle. In conclusion, this study has found that local macrophage proliferation plays an important role in the pathogenesis of granuloma formation.     

Development and heterogeneity of macrophages and their related cells through their differentiation pathways

Macrophages are a heterogeneous population differing In their site of location, morphology and function. They develop from hematopoletic stem cells originating In both fetal and bone marrow hematopolesls. In yolk sac and early hepatic hematopoiesls, primitive/fetal macrophages develop from hematopoletic stem cells, bypassing the stage of monocytic cells (monoblasts, promonocytes and monocytes), possess proliferatlve capacity and differentiate into resident macrophages in tissues in late ontogeny. Monocytic cells develop In hepatic hematopolesis after the development of primitive/ fetal macrophages, then move into the bone marrow in late ontogeny, forming a monocyte-derived macrophage population in tissues. Like monocytes, the monocyte-derived macrophages have no proliferative potential and are short-lived, whereas the resident macrophages are long-lived in tissue, possess proliferatlve capacity and can be sustained by self-renewal. In adult life, the bone marrow releases macrophage precursors (immature myeloid cells) and monocytes into peripheral blood, but normally not monoblasts or promono-cyts. The myeloid precursor cells migrate into tissues and differentiate into resident macrophages or related cells in situ due to macrophage differentiation or growth factors, such as M-CSF and GM-CSF, produced in situ and/or supplied Immorally. Monocytes, however, migrate into tissues in response to inflammatory stimuli and differentiate Into exudate macrophages. The distinct differentiation pathways of monocyte/macrophages, resident macrophages, other macrophage subpopulations, and macrophage-related cells are reviewed together with the heterogeneity of macrophage precursor cells.     

In vivo responses of macrophages and myofibroblasts in the healing following isoproterenol-induced myocardial injury in rats

To clarify the relation between macrophage and myofibroblast involvement in various myocardial diseases, the authors investigated the kinetics of these cells in the healing (scar tissue formation) following isoproterenol-induced myocardial injury in rats. Alphasmooth muscle actin (-SMA) expressing myofibroblasts were seen at the border of the affected area and appeared in the greatest numbers on days 3–7 post-injection, followed by a gradual decrease by day 35. The peak on day 3 was consistent with the timing of the highest proliferative activity of myofibroblasts. The number of ED1-positive macrophages began to increase as early as day 1, reaching a peak on day 3 within the injured myocardium. The expansion of EDI-positive macrophages preceded an increased number of -SMA-positive myofibroblasts suggesting that myofibroblast proliferation and activation may be mediated by factors released by ED1-positive mcrophages in response to myocardial injury. The number of ED2-positive tissue-fixed, resident macrophages gradually increased from day 3 post-injection, and peaked on day 14, but the number of ED2-positive macrophages was consistently fewer than that of ED1-positive macrophages during the 35 day-observation period after the injection. The labelling index of the ED2-positive cells was maximal on day 14, indicative of local proliferation of resident macrophages. In the healing process after myocardial injury, EDI-positive macrophages increase markedly in the early stages; ED2-positive macrophages appear later.     

Proliferation Kinetics of Rat Kupffer Cells after Partial Hepatectomy Immunohistochemical and UItrastructural Analysis

In an effort to settle the conflicting views on the proliferation kinetics of Kupffer cells (Kc), we performed 2/3 partial hepatectomy on rats injected with Pelikan ink. Using an anti-rat macrophage monoclonal antibody, ED 2, we evaluated the numerical changes in total, carbon-positive ED 2⁺ cells and carbon-negative ED 2⁺ cells in the portal and central area. We also analyzed the ultrastructure and peroxidase cytochemistry of various types of cells observed during regeneration. The total numbers of ED 2⁺ cells in the remaining liver increased rapidly from day 2 to 5, and the number of dividing ED 2⁺ cells reached a maximum on day 2. Thus, the numerical increase in ED 2⁺ cells corresponded to the division phase. In contrast, the carbon-labeling experiment showed a continuous increase of carbon negative ED 2⁺ cells from day 2 to 7. In the central area where division was less frequent, the proportion of carbon-positive cells decreased markedly to 50% on day 7, as against 97% in control rats. These findings suggest the possibility of an influx of carbon-negative Kc in addition to cell division. Ultrastructurally, the presence of carbon-negative "small Kc" and "immature Kc" with morphological features different from those of carbon-positive Kc was demonstrated. Such carbon-negative Kc with a high nucleus-to-cytoplasm ratio and rather few phagosomes, were not observed in control rats. Furthermore, we demonstrated two types of possible precursor cell, i.e. "transitional" forms between monocytes and Kc, and "immature macrophages". The former showed peroxidase activity in some lysosomes as well as in the rough endoplasmic reticulum and nuclear envelope. Our result indicated that the proliferation kinetics of Kc depend upon both local proliferation and influx.     

Kupffer cell restoration after partial hepatectomy is mainly driven by local cell proliferation in IL-6-dependent autocrine and paracrine manners

Kupffer cells (KCs), which are liver-resident macrophages, originate from the fetal yolk sac and represent one of the largest macrophage populations in the body. However, the current data on the origin of the cells that restore macrophages during liver injury and regeneration remain controversial. Here, we address the question of whether liver macrophage restoration results from circulating monocyte infiltration or local KC proliferation in regenerating livers after partial hepatectomy (PHx) and uncover the underlying mechanisms. By using several strains of genetically modified mice and performing immunohistochemical analyses, we demonstrated that local KC proliferation mainly contributed to the restoration of liver macrophages after PHx. Peak KC proliferation was impaired in Il6-knockout (KO) mice and restored after the administration of IL-6 protein, whereas KC proliferation was not affected in Il4-KO or Csf2-KO mice. The source of IL-6 was identified using hepatocyte- and myeloid-specific Il6-KO mice and the results revealed that both hepatocytes and myeloid cells contribute to IL-6 production after PHx. Moreover, peak KC proliferation was also impaired in myeloid-specific Il6 receptor-KO mice after PHx, suggesting that IL-6 signaling directly promotes KC proliferation. Studies using several inhibitors to block the IL-6 signaling pathway revealed that sirtuin 1 (SIRT1) contributed to IL-6-mediated KC proliferation in vitro. Genetic deletion of the Sirt1 gene in myeloid cells, including KCs, impaired KC proliferation after PHx. In conclusion, our data suggest that KC repopulation after PHx is mainly driven by local KC proliferation, which is dependent on IL-6 and SIRT1 activation in KCs.     

Hydroxysafflor yellow A attenuates ischemia/reperfusion-induced liver injury by suppressing macrophage activation

Hydroxysafflor yellow A (HSYA), a major constituent in the hydrophilic fraction of the safflower plant, can retard the progress of hepatic fibrosis. However, the anti-inflammatory properties and the underlying mechanisms of HSYA on I/R-induced acute liver injury are unknown. Inhibiting macrophage activation is a potential strategy to treat liver ischemia/reperfusion (I/R) injury. In this study, we investigated the therapeutic effect of HSYA on liver I/R injury and the direct effect of HSYA on macrophage activation following inflammatory conditions. The therapeutic effects of HSYA on I/R injury were tested in vivo using a mouse model of segmental (70%) hepatic ischemia. The mechanisms of HSYA were examined in vitro by evaluating migration and the cytokine expression profile of the macrophage cell line RAW264.7 exposed to acute hypoxia and reoxygenation (H/R). Results showed that mice pretreated with HSYA had reduced serum transaminase levels, attenuated inflammation and necrosis, reduced expression of inflammatory cytokines, and less macrophage recruitment following segmental hepatic ischemia. In vitro HSYA pretreated RAW264.7 macrophages displayed reduced migratory response and produced less inflammatory cytokines. In addition, HSYA pretreatment down-regulated the expression of matrix matalloproteinase-9 and reactive oxygen species, and inhibited NF-κB activation and P38 phosphorylation in RAW264.7 cells. Thus, these data suggest that HSYA can reduce I/R-induced acute liver injury by directly attenuating macrophage activation under inflammatory conditions.     

Macrophage Phenotype in Liver Injury and Repair

Macrophages hold a critical position in the pathogenesis of liver injury and repair, in which their infiltrations is regarded as a main feature for both acute and chronic liver diseases. It is noted that, based on the distinct phenotypes and origins, hepatic macrophages are capable of clearing pathogens, promoting/or inhibiting liver inflammation, while regulating liver fibrosis and fibrolysis through interplaying with hepatocytes and hepatic stellate cells (HSC) via releasing different types of pro‐ or anti‐inflammatory cytokines and growth factors. Macrophages are typically categorized into M1 or M2 phenotypes by adapting to local microenvironment during the progression of liver injury. In most occasions, M1 macrophages play a pro‐inflammatory role in liver injury, while M2 macrophages exert an anti‐inflammatory or pro‐fibrotic role during liver repair and fibrosis. In this review, we focused on the up‐to‐date information about the phenotypic and functional plasticity of the macrophages and discussed the detailed mechanisms through which the phenotypes and functions of macrophages are regulated in different stages of liver injury and repair. Moreover, their roles in determining the fate of liver diseases were also summarized. Finally, the macrophage‐targeted therapies against liver diseases were also be evaluated.     

Bone marrow-derived macrophage contributes to fibrosing steatohepatitis through activating hepatic stellate cells

The role of macrophages in fibrosing steatohepatitis is largely unclear. We characterized the origin and molecular mechanisms of macrophages and its targeted therapy of fibrosing steatohepatitis. Fibrosing steatohepatitis was established in Alms1 mutant (foz/foz) and C57BL/6J wildtype mice fed high-fat/high-cholesterol or methionine- and choline-deficient diet. Bone marrow transplantation was performed to track the macrophage origin in fibrosing steatohepatitis. Macrophages were depleted using liposomal clodronate. Primary macrophages were isolated from bone marrow for adoptive transfer into mice. We found that macrophage infiltration is induced in two mouse models of fibrosing steatohepatitis and human nonalcoholic steatohepatitis-fibrosis patients. Bone marrow-derived macrophages (BMMs) contribute to the hepatic macrophage accumulation in experimental fibrosing steatohepatitis. Depletion of hepatic BMMs by liposomal clodronate during liver injury attenuated fibrosing steatohepatitis, whilst BMMs depletion after liver injury delayed the regression of fibrosing steatohepatitis. The pro-fibrotic effect of macrophages was associated with reduced activation of hepatic stellate cells (HSCs), collagen deposition and hepatic expression of key pro-fibrotic factors (TIMP1, TIMP2, and TGFβ1) and endoplasmic reticulum stress markers (GRP78, IRE1α, and PDI). Conversely, adoptive transfer of BMMs significantly aggravated fibrosing steatohepatitis. Moreover, macrophage-conditioned medium directly promoted the phenotypic transition of primary quiescent HSCs to activated HSCs; it enhanced activation and proliferation but decreased apoptosis of HSC cell lines (LX-2 and HSC-T6). The effect of BMMs in promoting fibrosing steatohepatitis was mediated by inducing key pro-fibrosis factors and signaling pathways including cytokine/chemokine, TGFβ and complement cascade as assessed by cDNA expression array. Complement 3a receptor (C3ar1) was a predominant effector of macrophage mediated fibrosing steatohepatitis. Knockout of C3ar1 in mice blunted development of fibrosing steatohepatitis. In conclusion, BMMs promoted the progression of fibrosing steatohepatitis during injury, whereas macrophages reduced fibrosing steatohepatitis in the recovery phase of liver injury. Increasing anti-fibrotic macrophages and decreasing pro-fibrotic macrophages are promising approaches for fibrosing steatohepatitis. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Emergence of different macrophage populations in hepatic fibrosis following thioacetamide-induced acute hepatocyte injury in rats

Macrophages may play a role in fibrogenesis. The kinetics and distribution of different macrophage populations were investigated immunohistochemically in hepatic lesions following acute hepatocyte injury induced in F344 rats by a single injection of thioacetamide (TAA) (300 mg/kg body weight, intraperitoneally). Hepatocyte degeneration or necrosis induced by TAA occurred mainly in the perivenular areas of hepatic lobules as early as post-injection (PI) days 1 and 3; fibrotic lesion development began in the damaged areas on day 1, and peaked on day 5; thereafter (PI days 7 and 10), the fibrotic areas decreased and were replaced by regenerated hepatocytes on PI days 15 and 20, indicating a remodelling process. In this rat model, the number of macrophages reacting with ED1 antibody (specific for exudate macrophages), ED2 (recognizing cell membrane antigens of resident macrophages, including Kupffer cells) and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages and dendritic cells) began to increase on PI day 1, peaking on PI day 3. The numbers gradually decreased on PI days 5 and 7; however, the statistically significant increase was maintained in respect of ED1-positive cells up to PI day 20, whereas no significant increase in ED2- and OX6-positive cells remained from PI day 10 onwards. Interestingly, of the ED1-, ED2- and OX6-positive cells, the OX6-positive cells were the least numerous. ED1- and OX6-positive cells appeared exclusively in the injured perivenular areas, whereas ED2-positive cells were present mainly in the mid-zonal areas and in smaller numbers in the perivenular areas. These findings indicated differences in kinetics and distribution between macrophage populations appearing in hepatic fibrosis. In addition, RT-PCR revealed that mRNA expression of osteopontin, a factor for induction and maintenance of macrophages in inflammation, was markedly increased on PI days 5, 7 and 10, suggesting a role in the pathogenesis of hepatic fibrosis.     

Immunohistochemical Detection of Myeloperoxidase and Its Oxidation Products in Kupffer Cells of Human Liver

Oxidative damage to tissue proteins has been implicated in the pathogenesis of liver disease, but the mechanisms that promote oxidation in vivo are unclear. Hydrogen peroxide is transformed into an array of potentially damaging reactants by the heme protein myeloperoxidase. This proinflammatory enzyme is expressed by circulating neutrophils and monocytes but is generally thought to be absent from tissue macrophages. To determine whether myeloperoxidase is present in Kupffer cells, the fixed-tissue macrophages of liver, Western blot analysis, and immunohistochemistry were performed. Two different antibodies monospecific for myeloperoxidase identified a 60-kd protein, the predicted molecular mass of myeloperoxidase, in human liver extracts. Immunostaining detected the enzyme in sinusoidal lining cells of normal and diseased human livers. Immunofluorescence confocal microscopy demonstrated co-localization of myeloperoxidase and CD68, a monocyte/macrophage marker, in sinusoidal lining cells. Numerous myeloperoxidase-expressing cells were also evident in the fibrous septa of cirrhotic livers. Immunostaining with an antibody to proteins modified by hypochlorous acid, a characteristic product of the enzyme, indicated that myeloperoxidase is enzymatically active in cases of acute liver injury and cirrhosis. These findings identify myeloperoxidase as a component of human Kupffer cells. Oxidative damage resulting from the action of myeloperoxidase may contribute to acute liver injury and hepatic fibrogenesis.     

Macrophage populations and apoptotic cells in the liver before spontaneous hepatitis in Long-Evans Cinnamon (LEC) rats

The inbred mutant strains of Long-Evans Cinnamon (LEC) rats spontaneously develops acute hepatitis as a result of abnormal copper accumulation, followed by chronic hepatitis, cholangiofibrosis and hepatocellular carcinoma. To shed some light on the role of macrophages in the liver failure, immunohistochemical methods were used to investigate the kinetics of macrophage populations in the liver of male LEC rats, in relation to the appearance of myofibroblastic cells and hepatocyte apoptosis. Rats examined at 24 weeks of age and moribund rats killed at 22-25 weeks of age had increased serum concentrations of aspartate aminotransferase and alanine aminotransferase, with jaundice and histological changes indicative of hepatic failure, whereas rats examined at 8, 12, 16 or 20 weeks old showed no such abnormal findings. Immunolabelling with ED1 (a monoclonal antibody recognizing rat macrophages) and ED2 (a monoclonal antibody specific for rat resident macrophages) revealed that numbers of blood monocyte-derived macrophages and Kupffer cells began to increase markedly at 16 weeks of age (before the onset of hepatitis). However, alpha-smooth muscle actin (SMA)-positive myofibroblastic cells (modulated perisinusoidal cells) and hepatocyte apoptosis, demonstrable by the TUNEL method, were rarely seen at 8, 12, 16, 20 or 24 weeks. There was no close relationship between macrophage expansion and the appearance of myofibroblastic cells or hepatocyte apoptosis. In moribund rats, only a few SMA-positive cells were seen in the periportal zones; hepatocytes undergoing apoptosis increased in number, and macrophages engulfing apoptotic bodies were observed occasionally, suggesting that apoptosis was related to hepatic failure as an early event. In addition, immunohistochemical examination demonstrated abnormal deposits of laminin along the sinusoids from 20 weeks, as an initial extracellular matrix protein in LEC rat livers.     

Macrophage production of basic fibroblast growth factor in the fibroproliferative disorder of alveolar fibrosis after lung injury.

In organ repair following injury, macrophages accumulate and granulation tissue, comprised of fibroblasts and endothelial cells, develops in the injured area. Basic fibroblast growth factor (bFGF), a potent stimulator of fibroblast and endothelial cell growth, has been linked to the fibroproliferative process. Macrophages are thought to play a central role in the fibroproliferative response, and prior studies indicate that they produce bFGF. Whereas it is plausible that macrophages produce bFGF in a fibroproliferative process, currently no data exists that directly identifies the macrophage as a source of bFGF in a fibroproliferative disorder. We used the model of acute intraalveolar granulation tissue formation following lung injury to determine if the macrophage was a cellular source of bFGF in a naturally occurring fibroproliferative process. To examine this hypothesis, patients with severe acute lung injury underwent bronchoalveolar lavage during the phase of lung repair. Polymerase chain reaction and Northern analysis of macrophage RNA revealed the presence of two species of bFGF messenger RNA (4.4 kb and 1.9 kb). Metabolic labeling studies of recovered macrophages revealed a newly synthesized 18-kd protein with antigenic similarity to bFGF. Immunohistochemical evaluation of lung tissue from patients who died following acute lung injury, showed numerous bFGF immunoreactive macrophages present within airspaces containing fibroblastic and vascular tissue proliferation. This investigation has identified the alveolar macrophage as a cellular source of bFGF in the fibroproliferative disorder of intraalveolar fibrosis following acute lung injury.     

Neurons and neuroblastoma as a source of macrophage colony-stimulating factor.

Accumulation of macrophages in brain tissue as observed in nervous system injury may be due to local production of hematopoietic colony-stimulating factors (CSF). The present work shows human neuroblastoma cells and murine neurons, namely granule cells of the cerebellum, to produce macrophage (M)-CSF which guides expansion and differentiation of macrophage lineage cells. The mRNA-encoding M-CSF but not the respective protein is present in mouse brain including cerebellum. Neither granulocyte M-CSF nor IL-3 is produced by cerebellar neurons or neuroblastoma. By their production of M-CSF, neurons may regulate the macrophage response and lead to local expansion and enhanced function of macrophages in inflammatory diseases of the central nervous system.     

Distinctive roles of neutrophils and monocytes in anti-thy-1 nephritis

Anti-Thy-1.1 glomerulonephritis as an experimental model for mesangial proliferative glomerulonephritis was induced in Wistar rats by a single injection of monoclonal IgG2a-anti-Thy-1.1 antibody (ER4G). This transient model is complement-mediated and leads to mesangial-cell (MC) lysis followed by MC proliferation, glomerular microaneurysm formation, glomerular influx of polymorphonuclear leukocytes (PMNs) and macrophages, proteinuria, and hematuria. In this study we investigated the distinctive roles of infiltrating PMNs or monocytes/macrophages by treating rats with an antibody against rat integrin CD11b/CD18 (ED7) or by depletion of monocytes with multilamellar clodronate liposomes, respectively. ED7 administration resulted in reduction of the influx of PMNs in glomeruli during the first 6 days after induction of Thy-1.1 nephritis, whereas treatment with an isotype-matched irrelevant antibody (PEN9) or with phosphate-buffered saline had no effect on macrophage influx. Increased glomerular C3 and C6 deposition on days 1 and 3 was seen in the ED7-treated rats but not seen in the control groups. In addition, the ED7-treated group showed an increased number of aneurysmatic glomeruli and more severe hematuria. Monocyte/macrophage depletion led to a significant reduction of mesangial matrix expansion, although mesangial proliferation, proteinuria, and hematuria remained unaltered. These results, together with the known effects of PMN-derived enzymes on C3 cleavage, suggest that a reduction in the influx of PMNs results in sparing of C3 and consequently of more complement activation in the glomerulus with increased complement-mediated damage. Our data indicate that infiltrating PMNs and monocytes/macrophages play distinctive roles during inflammation in this model of MC glomerulonephritis.