Anti-CD11b/CD18 antibodies reduce inflammation in acute colitis in rats.

The influx of monocytes and neutrophils into the inflamed tissue could be an important aspect in the pathogenesis of inflammatory bowel disease (IBD). A membrane protein involved in the monocyte/neutrophil adherence to endothelium is CD11b/CD18 or alpha M beta 2 (complement receptor type 3 = CR3). In the present study the role of CD11b/CD18 in experimental IBD was studied by treatment with ED7 and OX42, two MoAbs against CD11b/CD18. Colitis was induced in rats by a single, rectal administration of 30 mg 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol 30%. Two hours before and 3 days after induction of colitis, the animals were given an i.v. dose of 0.5 mg of either ED7 or OX42 in 1 ml PBS. Controls received PBS or an irrelevant MoAb. Four days after the last treatment with the antibodies, the rats were killed, and macroscopic damage scores of the colon were determined. Macrophages and granulocytes were studied by immunohistochemistry and quantified by Interaktives Bild Analysen System (IBAS), and myeloperoxidase (MPO) activity in colonic tissue was measured. After treatment with ED7 and OX42 the mean damage score of the colon was reduced from 4.2 in IBD animals to 1.0 and 1.3, respectively. Smaller areas of ulcerations and a decrease in the number of ulcerations were observed compared with PBS-treated rats. Furthermore, the amount of infiltrating monocytes and leucocytes in the submucosa was enormously reduced, as well as MPO activity in the colonic tissue. These results show that treatment with MoAbs against CD11b/CD18 reduces clinical signs of experimental IBD in rats by a partial blockade of infiltrating macrophages and granulocytes.

Macrophage depletion in the rat after intraperitoneal administration of liposome-encapsulated clodronate: depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund's adjuvant.

The purpose of this study was to develop a method for the depletion of macrophages from the peritoneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with liposome-encapsulated clodronate (dichloromethylene bisphosphonate: Cl2MBP-liposomes). This treatment resulted in complete elimination of mature tissue macrophages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were completely depleted, few ED1-positive cells remained and repopulation of ED1-positive cells was faster. The treatment further depleted macrophages from the spleen, especially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of Cl2MBP-liposomes considerably accelerated repopulation in the omentum. The protocol described might be used to investigate the contribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimination of intestinal tumours.

Non-lymphoid and lymphoid cells in acute, chronic and relapsing experimental colitis.

In rodents, intracolonic administration of ethanol 30% induces an acute colitis, while administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS) in ethanol induces a longer lasting colitis. In the acute and chronic stages of experimental colitis, lymphoid and non-lymphoid cells were studied in the colon by immunohistochemistry. During the acute inflammation a high damage score of the colon was observed, which was related to an increase in the number of macrophages and granulocytes. Also a change in distributional patterns of macrophage subpopulations was found. The chronic stage of TNBS-ethanol-induced colitis was characterized by an increase in the number of lymphocytes, especially T cells. These data suggest that macrophages and granulocytes are important in the acute phase of experimental colitis, while lymphocytes play a pivotal role in the chronic stage. As most inflammatory bowel disease (IBD) patients have relapses during the chronic disease, we attempted to induce a relapse during experimental colitis by giving a second i.p. or s.c. dose of TNBS. This resulted in increased damage scores of the colon, new areas of ulceration and a further increase in macrophage numbers. No effect on the number of granulocytes was seen. These results indicate that it is possible to mimic relapses in experimental colitis by a second administration of TNBS, and suggest that the rats had been sensitized by the first dose of TNBS, given into the colon.

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 His14 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 CC531 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.

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat.

The development of macrophage subpopulations dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gut-associated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.

Macrophage subsets in the rat gut: an immunohistochemical and enzyme-histochemical study.

Macrophages' subsets including dendritic cells were characterized in situ and in cell suspensions of Peyer's patches, intestinal villi, and mesenteric lymph nodes using monoclonal antibodies, lectins, and enzyme-histochemistry. By combined labelling procedures, it was found that Peyer's patches comprise dendritic cells in addition to 2 types of macrophages; dendritic cells were not observed among the various macrophage-like cells in the lamina propria of the villi. The lectins Dolichos biflorus and Griffonia simplicifolia I agglutinin appeared to be useful for discriminating between several subtypes of macrophages.

The ontogenetic development of macrophage subpopulations and Ia-positive non-lymphoid cells in gut-associated lymphoid tissue of the rat.

The ontogenetic development of macrophage subpopulations and Ia-positive non-lymphoid cells was studied in gut-associated tissue in fetal and neonatal Wistar rats. A two-step immunoperoxidase method was carried out on cryostat sections, a panel of monoclonal antibodies being applied and aimed specifically at rat macrophages (ED1, ED2 and ED3) and at Ia antigen (Ox4). The first ED1-positive macrophages appeared in the liver on Day 15 (gestational age), and they did not express Ia. In developing mesenteric lymph nodes and in the gut wall, macrophages were found for the first time on Day 17 and 18, respectively, of gestation. These early macrophages were also ED1-positive. Until birth, ED2 recognized few cells in the gut-associated tissue; on the day of birth this subpopulation showed a sudden and considerable increase. The distribution pattern of ED3-positive macrophages appeared to be the same in fetal and in adult rats; it was confined to lymph nodes and Peyer's patches. Ia-positive non-lymphoid cells appeared in the abdomen early in ontogeny. The first Ia-positive cells displayed dendritic features and were found on Day 15 of fetal life in the mesenchymal tissue between intestinal loops. A few days later, many Ia-positive cells with a dendritic appearance were demonstrable in the gut wall and in developing mesenteric lymph nodes. Their number increased rapidly during the following days. Based on these results, the existence of two differentiation lines for dendritic cells and classical macrophages is discussed. The function of early Ia-positive cells in the abdomen is suggested as not being an antigen-presenting one.

Lymph node involvement in a humoral immune response.

Mice were primed subcutaneously in the hind footpads with horseradish peroxidase (HRP) and boosted intravenously 10 weeks later. The appearance of cells with cytoplasmic anti-HRP antibody was studied in several lymph nodes. It appeared that following intravenous boosting antibody-forming cells appeared in significant numbers in the popliteal, the lumbar, and (in some animals) the sciatic lymph nodes exclusively. In the other lymph nodes examined specific antibody-forming cells were observed only occasionally. By subcutaneous injection of Evans blue in the hind footpads it was shown that the subcutis of the hind footpads of these animals is drained by the popliteal lymph nodes, the lumbar lymph nodes, and (to a lesser degree) the sciatic lymph nodes. The presence of trapped immune complexes within lymph node germinal centers was confined to these three nodes. Based on these findings, it is concluded that specific antibody-forming cells during the secondary response in these mice are induced exclusively in the lymph nodes draining the site of primary immunization.