The Role of the Macrophage in Wound Repair a Study with Hydrocortisone and Antinwcrophage Serum

The Role of the Macrophage in Wound Repair A Study with Hydrocortisone and Antinwcrophage Serum S. J. Leibovich, PhD and Russell Ross, PhD The role of the monocyte/macrophage in wound repair has been investigated by studying the healing process in wounds depleted of this cell and/or its phagocytic activity. Hydrocorfisone acetate (0.6 mg/g body weight) administered as a subcutaneous depot was used to induce a prolonged monocytopenia in guinea pigs, and antimacrophage serum (AMS) was used for local elimination of tissue macrophages. In vitro, in the presence of complement, macrophages are rapidly lysed and killed by AMS. In the absnce of complement, AMS is not cytotoxic but potently inhibits adherence to and phagocytosis of opsonized erydtcytes by macrophages. AMS titers were obtained by observation of adherence and phagocytosis of opsonized erythrocytes in serial dilutions of AMS. Six groups of animals were studied: a) untreated animals, b) animals receiving daily subcutaneous injections of normal rabbit serum (NRS) around each wound, c) animals receiving daily subcutaneous AMS around each wound, d) animals receiving systemic hydrocortisone, e) animals receiving systemic hydrocortisone and daily injections of NBS around each wound, and f) animals receiving systemic hydrocortisone and daily AMS around each wound. Wounds consisted of a series of six linear incisions in the dorsal skin. Subcutaneous AMS alone had no effect on the number of circu- lating monocytes, nor was there any observable effect on the number or the phagocytic ability of wound macrophages. Fibrosis in these wounds was unaffected. Systemic hydrocortisone inducd a prolonged monocytopenia. The macrophage level in the wounds of these monocytopenic animals was reduced to approximately one-third that of controls; the phagocytic activity of the monocytes/macrophages that did appear in these wounds was, however, smilar to that of controls. Some inhibition of wound debridment was observed in these wounds, but fibrosis was virtually unaffected. Collagen synthesis, as judged morphometrically, was similar to that of control wounds at all stages of repair. Conjoint systemic hydrocortisone and subcutaneous AMS around each wound resulted in the almost complete dis- appearance of macrophages from the wounds. Wound fibrin levels were elevated, and clearance of fibrin, neutrophils, erythrocytes and other miscellaneous debris from these wounds was delayed. Fibroblasts, which in control wounds first appear by 3 days postwounding and reach maximal levels by day 5, did not appear in these wounds until day 5, and their subsequent rate of proliferation was slower than that of controls. Seven and 10 day wounds appeared immature both in ter of the degree of debridement, and extent of fibrosis. These studies indicate that, in the repair process, the principle cell type responsible for wound debridement is the macrophage. In addition, the macrophage may be required to stimulate fibroblast proliferation in some as yet unidentified manner. (Am J Pathol 78:71- 100, 1975). From the Department of Pathology, University of Washington School of Medicine, Seattle, Washington. Supported in part by Grant ANM-13970 from the US Public Health Service. Accepted for publication September 10, 1974. Address reprint requests to Dr. Russell Ross, Department of Pathology, University of Washington, School of Medicine, Seattle, WA 98195. 71 72 LEIBOVICH AND ROSS American Journal of Pathology THE INFLAMMATORY RESPONSE resulting from tissue injury after wounding is characterized by a relatively rapid accumulation of numerous polymorphonuclear neutrophilic leukocytes and macrophages at the site of injury.'-3 While both of these cell types begin to emigrate from the blood vessels adjacent to the wound simultaneously, the neutrophils reach a maximal level in the wound by the first 2 days and then decrease in number, while macrophages reach maximal levels somewhat later, after approximately 3 days, by which time they are the principle phagocytic cells within the wound. Subsequently, immature fibroblasts, newly formed collagen fibrils and numerous capil- laries appear within the wounds.1-3 While the general sequence of events which takes place during wound repair has been well characterized, the relationships between each of the various cellular and humoral components of the inflammatory response, and their relationship to the stimulation of fibroblast proliferation and collagen formation are not well understood. A recent investigation from this laboratory was concerned with the role played by the neutrophil in wound repair.45 Using antineutrophil serum to induce a neutropenia in guinea pigs, it was possible to study the healing process in the absence of these cells. In the absence of gross infection, repair in the neutropenic animals proceeded in a manner identical to that in normal animals.5 It was concluded that the presence of neutrophils in the wounds, although critical in the presence of infection, was not an essential antecedent to fibrogenesis, as had been sug- gested by previous workers.6-8 Also, the presence of neutrophils was not necessary to stimulate the migration of monocytes into the wounds. This study is concerned with the role played by the monocyte/ macrophage in wound repair. Our approach was to attempt to elimi- nate the macrophage from healing wounds, and to study the changes in the course of wound repair resulting from either depletion of these cells and/or impairment of their ability to be phagocytic. Materials and Methods Experimental Animals Rockefeller strain guinea pigs were used both as the source of macrophages for the preparation of AMS, and for the studies of wound repair. Preparation of Antimacrophage Serum Peritoneal exudates were obtained from 400- to 500-g guinea pigs 4 to 5 days after intraperitoneal injection of 10 ml sterile mineral oil (Invenex Muri-Lube "Heavy"). Animals were killed by ether inhalation, and 50 ml sterile isotonic phosphate-buffered saline (PBS) (containing heparin, 1000 USP units/5 ml) was injected into the peritoneal cavity. Following gentle kneading, the cell-rich Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 73 January 1975 fluid was removed. Differential cell counts were performed on Wright's-Giemsa- stained smears of the cell suspension. The exudates generally contained better than 95% macrophages, with small numbers of lymphocytes, neutrophils, erythrocytes and occasional mesothelial cells. The cell suspension was centrifuged at 100 g for 5 minutes and then washed three times with PBS. The cells were counted and finally suspended in Hanks' solution at a concentration of 2 x 108 cells/mL Three New Zealand strain adult male rabbits were injected intraveneously via the marginal ear vein with 2 ml of the macrphage cell-suspension. Two weeks later the rabbits were given a similar injection with an equal number of cells. One week after the second injection, the rabbits were exsanguinated by cardiac puncture, and serum prepared from the whole blood. The sera from the 3 animals were pooled and heat inactivated at 56 C for 30 minutes. After titration and absorption as descnbed below, the AMS was sterilized by filtration and stored at -20 C until used. Normal rabbit serum (NRS) for control experiments was collected from untreated rabbits from the same stock used for AMS preparation. Control serum was processed in an identical fashion to that of the AMS. Intravenous injection of macrophages suspended in Hanks' solution provided antisera with improved titers of AMS having less crs reactivity with other cell types than did other methods of raising the antibody, such as subcutaneous injection with Freund's adjuvant. tration of AntiSmophg m It has been demonstrated previously that AMS inhibits phagocytosis of opsonized erythrocytes by macrophages in vitro."'12 We have utilized this property as the basis of an assay for AMS activity. 'Macrophages were prepared from mineral-oil-stimulated peritoneal exudates; 1.2 x 106 cells were cultured in each of several small petri dishes (Falcon, 3 cm) using Waymouth's medium containing 10% fetal calf serum. A square glass cover slip was placed in each dish prior to addition of the macrophages. The ceHls rapidly adhere to the cover slip. After 1 hour, nonadherent cells were removed and replaced with fresh medium. Duplicate dishes containing several dilutions of AMS from 1:10 to 1:1280 were prepared. Control dishes with serially diluted NRS were also prepared. Sheep red blood ceHls (RBC) were sedimented at lOOg for 10 minutes and washed three times with PBS. Antisheep cell hemolysin (BBL Bio Quest) was added to the RBC suspension to a dilution of 1:400, and the suspension was incubated at 37 C for 20 minutes. The cells were sedimented at lOOg for 5 minutes and washed three times with PBS to remove excess antibody; 0.2 ml RBC were added to each dish of cultured macrophages containing serially diluted AMS or NRS. The cells were then incubated at 37 C for 2 hours. At the end of this incubation period the culture medium was removed, and the cells were washed briefly with Way- mouth's medium (without serum). Cover slips, removed and mixed for 10 minutes with absolute ethanol followed by staining with Wright's Giemsa, were examined in the light microscope. Serial d[lutions of the AMS demonstrated a concentration at which adherence and phogocytosis occurs, above which normal adherence and phagocytosis of opsonized erythrocytes is observable and below which neither adherence nor phagocytosis is present. This method provides a rapid and simple assay of the activity of each batch of AMS (see Note Added in Proof). Absorption of Antimacrophage Serum Serum Antimacrophage serum was absorbed with normal guinea pig serum (0.05 ml/ml ANMS) for 1 hour at 37 C in a shaking water bath. Tle serum was then centrifuged 74 LEIBOVICH AND ROSS American Journal of Pathology at 30,000g for 30 minutes at 4 C, and the supernatant collected. The absorbed serum gave no precipitin line, as determined in double-diffusion analysis in agar plates (immunoplate, Hyland Labs, Los Angeles, Calif).

Load more