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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 /macrophage in wound repair has been investigated by studying the process in wounds depleted of this cell and/or its phagocytic activity. Hydrocorfisone acetate (0.6 mg/g body weight) administered as a sub- cutaneous 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 of opsonized erydtcytes by macrophages. AMS titers were obtained by observation of adherence and phago- 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 hydro- cortisone, 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 . Subcutaneous AMS alone had no effect on the number of circu- lating , nor was there any observable effect on the number or the phagocytic ability of wound macrophages. 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. 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, , erythrocytes and other miscellaneous debris from these wounds was delayed. , 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 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 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, im- mature 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 pro- liferation and collagen formation are not well understood. A recent investigation from this laboratory was concerned with the role played by the in wound repair.45 Using antineutrophil serum to induce a 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 infec- tion, 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 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 , 1000 USP units/5 ml) was injected into the . 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 , neutrophils, erythro- cytes 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 un- treated 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 , such as subcutaneous injection with Freund's adjuvant. tration of AntiSmophg m It has been demonstrated previously that AMS inhibits phagocytosis of opson- ized 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 (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).

Erythrocytes Guinea pig erythrocytes obtained by cardiac puncture from guinea pigs were washed 3 times and suspended in PBS. AMS was absorbed twice with 5 ml packed RBC/25 ml AMS. This was sufficient to remove hemagglutinating .

Lymphocytes Lymphocytes were prepared from guinea pig cervical, axillary and mesenteric lymph nodes as described by Henney.13 Lymph nodes were homogenized gently in PBS using a ground glass homogenizer. The homogenate was immediately centrifuged at 1500 rev/min for 5 minutes, and the supematant discarded. The sediment was resuspended in PBS and centrifuged at 900 rev/min for 30 seconds and the sediment discarded. The supernatant cells were then centrifuged at 1500 rev/min for 5 minutes and washed twice with PBS. Lymphocytes thus prepared were suspended at a concentration of 2 x 108 cells/ml in Ringer's solution. A 30-ml portion of AMS was absorbed twice with 2 ml of suspension. This reduced lymphocyte agglutinating titers to very low levels.

Neutrophils Neutrophils were prepared from guinea pig peritoneal exudates 18 hours after intraperitoneal administration of 40 ml of sterile 3% protease peptone (Difco Labs, Detroit, Mich) as described by Humphrey.14 fluid was removed, centri- fuged at 1000 rev/min and resuspended in PBS. The cells were washed twice with PBS and suspended finally at a concentration of 2 x 108 cells/ml; 30 ml AMS was absorbed twice with 2 ml of such concentrated neutrophils. Blood Leukocyte Counts The total leukocyte count of each animal was performed in duplicate for each of two samples from peripheral ear vein blood, from which the mean was calculated. Differential cell counts were performed on 400 leukocytes from at least two different blood smears fixed in absolute ethanol and stained with Wright's stain. From the total leukocyte and the differential cell count values, the absolute number of cells for each leukocyte category was determined.

Experimental Design and Wounding Glucocorticosteroid Administration The glucocorticosteroid preparation used in this study was hydrocortisone acetate as an emulsion in a vehicle (Hydrocortone, Merk, Sharpe & Dohme). The preparation was used in the commercially available form or was concentrated by centrifugation and resuspension so that the volume for injection was 1 ml. The hydrocortisone acetate was injected subcutaneously in the nuchal region. Vehicle preparations (containing 0.5% carboxymethylcellulose, 0.9% benzvl alcohol and 0.4% polysorbate 80 in saline) were used as controls and were found to have no effect on blood leukocyte levels. The hydrocortisone was administered as three separate doses, the first injection 3 days prior to wounding, the second on the day of wounding, and the final injection 4 days after wounding. The total dose admin- Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 75 January 1975

istered was equivalent to that used by Thomson and Van Furth 9.10 in mice, ie, 0.6 mg/g body weight. This dosage was found to give a similar reduction in guinea pig circulating monocyte levels as these workers found in mice (see Text-figure 1).

Antiserum Administration At the time of wounding, 0.5 ml NRS or AMS was placed directly in the wounds. Subsequently, AMS or NRS was injected subcutaneously locally around wounds. Four injections of 0.15 ml serm were administered per wound, at the wound apices, with a 26G, W-inch sterile needle. Wounding Experiments Guinea pigs weighing 350 to 400 g were used for all wounding experiments. Six experimental groups received different treatment of steroids and serum as follows: group 1, untreated; group 2, NRS alone; group 3, AMS alone; group 4, steroid alone; group 5, NRS and steroid; and group 6, AMS and steroid. Where steroid was administered, the first steroid injection was given 3 days prior to wounding, as described earlier. Animals were wounded as follows: the guinea pigs were lightly anesthetized with ether. The hair was shaved from their backs, and the skin was carefilly cleansed with 70% ethanol. A series of six linear incisions were made with a scalpel in the dorsal skin. Each incision was approximately 1.5 cm long, and extended through the panniculus camosus. Each animal was caged separately throughout the course of the experiment. They had access to a standard guinea pig pellet diet containing ascorbic acid (Ralston Purina, St. Louis, Mo) and water ad libitum. With the animals under ether anesthesia, wounds were removed at 1, 2, 3, 5, 7 and 10 days after wounding, by excision of an ellipse of skin containing the initiaJ incision. The biopsy sites were closed with wound clips.

Preparation of Wounds for Micmscop Ught Microscopy and Morphometry Immediately after excision the ellipse of skin containing the original wound was halved transversely and one-half of the wound was fixed with 10% neutral buffered formalin and embedded in paraffin. These embedded wounds were sec- tioned and stained with hematoxylin and eosin, phosphotungstic acid hemotoxylin, Van Giesons, and Gomori's trichrome stains. The remaining half of each wound was fixed with a one-half-strength modification of Karnovsky's glutaraldehyde/paraformaldehyde mixture.15 Tle tissue was im- mersed in fixative on a sheet of dental wax; using razor blades, slices of tissue, approximately 1 to 2 mm in thickness were cut so that each contained a cross section of the original wound. Fixation was continued for 4 hours at room tempera- hire. Tle tissue was then washed overnight at 4 C in 0.1 M cacodylate buffer containing 7.5% sucrose (pH 7.1) and postfixed for 1 hour at 4 C in 2% buffered osmium tetroxide. The tissue was then washed, stained en bloc with 0.5% uranyl acetate in 0.2 M s-collidine buffer (pH 6.1) at room temperature for 1 hour, dehydrated through graded alcohols and embedded in Epon. The wound slices were oriented in the blocks to obtain cross sections of each wound. For quantitation of wound components, 1i- sections from Epon blocks were routinely stained with basic fuchsin and alkalinized methylene blue,"6 and examined in the light microscope. Utilizing the I1i sections for orientation, the blocks were trimmed to the desired area for thin sectioning for electron microscopy. The thin 76 LEIBOVICH AND ROSS American Journal of Pathology sections were placed on carbon-coated grids, stained with uranyl acetate and lead citrate and examined in an AEI 801 electron microscope. Quantitative determinations of the relative volumes of the various cellular and extracellular and humoral components present in the wounds at various stages of repair were made using a stereologic method.'7 One-micron Epon sections stained with basic fuchsin-methylene blue were counted using a Zeiss Photoscope (Carl Zeiss, NY) at high dry magnification ( x 1600) with a 10-mm-square net reticule lined in 0.5-mm intervals (American Optical Corp, Buffalo, NY). Structures recorded included neutrophils, monocytes and macrophages, fibrin, red blood cells, fibroblasts, collagen, "space" and others (includes hair, fat, granulo- cytes other than neutrophils, unidentifiable cells, etc.). The category designated as space is that area not containing discrete structures, and it is thought to repre- sent fluid exudate containing plasma proteins, in addition to arising in part, from tissue processing artifact. The category designated as "other" made up a very small proportion of the total counts, and has been omitted from the tables. Sections of wounds from the various time periods were counted from at least 3 animals/experimental group. Sections from at least two blocks within a single wound (physically separated by at least 1 to 2 mm in vivo) were counted, and at least two sections per block were counted. Mean counts within each group were compared statistically within each category with a t-test. The accuracy of the counting technic was checked utilizing serial 1-y sections from within a single wound block. Four sections separated by about 20-[t intervals were stained and counted. Good internal agreement was found among these four sections, indicating that a high degree of reproducibility could be expected. Duplicate counts of the same section on different days also gave close agreement. Results Titration and In Vitro Effects of Antimacrophage Serum The procedure used in these investigations for titration of AMS by direct observation of adherence and phagocytosis of opsonized erythro- cytes by macrophages in the presence of serial dilutions of AMS pro- vides a rapid and convenient method for assaying the potency of various batches of antiserum. Titers of 1: 160 and greater were readily obtained using the procedure for raising AMS described above. After absorption with serum, erythrocytes, lymphocytes and neutrophils, small losses in activity were observed. Batches of AMS with final activity of less than 1:80 were not used in this study. In vitro effects of AMS are discussed in a separate publication." Briefly, in the absence of complement, AMS is not cytotoxic to macroph- ages, although characteristic morphologic changes do occur. Phagocy- tosis is, however, potently inhibited. In the presence of complement, macrophages are rapidly lysed and killed by AMS.

Hematologic Response to Hydrocortisone Administration Effects of Steroids Text figure 1 shows the absolute peripheral blood monocyte count, Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 77 January 1975

240

200

Z 160

120-

> 80-

40

0 2 4 6 8 10 12 14 16 Time (days) TEXr-Fsc 1-Peripheral blood monoyte for gluacrticoid-treated guinea pigs. Tbhe steroid was i subcutneously as an insoluble depot, using three separate injections on days 0, 3 and 7 (awTow). Total dosage was 0.6 mg/g body weight. following hydrocortisone injection. As Thomson and Van Furth demon- strated in mice, this dosage resulted in almost complete disappearance of monocytes from the peripheral blood.9l'0 The effect was maximal 3 days after injection, although a rapid drop in monocyte levels occurred within the first 12 hours. After 9 to 10 days, the level of monocytes began to rise gradually but by day 15 was still considerably lower than that in the control animals. A reduction in the level of circulating lymphocytes was also observed, although this effect was not as marked as the drop in monocyte levels. Lymphocytes were reduced to about 15% of control levels and after 9 or 10 days gradually increased in level. Neutrophil levels were un- affected by hydrocortisone for the first 3 days, but thereafter they grad- ually increased, until by 15 days after steroid administration the neu- trophil level was six to eight times higher than the controls.

Effect of Subcutaneous Normal Rabbit Serum and Antimacrophage Serum Both NRS and AMS caused a gradual increase in blood neutrophil levels to about four times normal levels after 15 days. Lymphocyte levels remained basically constant throughout the experiment in both groups, although by day 10 slight increases were observed. An initial drop in monocyte levels, of about 40%, occurred in both NRS and AMS 78 LEIBOVICH AND ROSS American Journal of Pathology groups, but this drop was not maintained, and by day 3 essentially normal levels were found that were maintained throughout the course of the experiment.

Effect of Steroids and Normal Rabbit Serum or Antimacrophage Serum Peripheral blood cell counts in these groups of animals were essen- tially the same as in animals treated with steroid alone.

Light Microscopy of Wound Repair Inflammatory Phase Control Wounds-Normal Rabbit Serum Alone and Untreated Wounds (Groups 1 and 2). Wounding resulted in initial hemorrhage and formation of a fibrin clot. Beneath the resultant scab of 24 hour control wounds, the interstices of the fibrinous clot were filled with numerous erythrocytes, neutrophils and monocytes/macrophages, and spaces presumably occupied by extravasated serum. Some of the macrophages at this stage showed marked evidence of phagocytic ac- tivity. In the region at the base of the scab, a dense demarcating layer of inflammatory cells was generally observed during the first 3 days. Neutrophils constituted the predominant cell type in this area, although some macrophages were also present. At 48 hours, approximately 18% of the total area of the control wounds was occupied by neutrophils, as opposed to 11% occupied by macrophages (See Figures 2 and 3). By 3 days, the number of neutrophils had decreased in relation to the num- ber of monocytes, with neutrophils occupying 7.5%, and monocytes about 15% of the total wound volume. At 3 days the macrophages were dis- tributed throughout the wound area and were actively phagocytic, con- taining numerous vacuoles with ingested material. Fibrin and red blood cells were still present at 3 days although fibrin levels had markedly decreased. Antimacrophage Serum Wounds (Group 3). No marked differences in were observed between AMS and NRS wounds. Neither the number of macrophages in the wounds, nor their phagocytic ability as judged by the presence of vacuoles and ingested material appeared to be affected by the AMS. Steroid Alone and Steroid/NRS Wounds (Groups 4 and 5). In these wounds, the area occupied by monocytes was reduced to approximately one-third that of the control wounds. The phagocytic ability of the macrophages, however, appeared to be unimpaired, since they con- Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 79 January 1975 tained phagocytic vacuoles, ingested neutrophils, RBCs and other miscellaneous debris. Wide variations in the patterns of fibrin deposition were observed, as in controls; however, the volume of the wounds oc- cupied by fibrin was somewhat increased (Text-figure 3). The number of neutrophils in these wounds was the same as in the control wounds. The principle effect of hydrocortisone, therefore, appeared to be a de- crease in the level of macrophages in the wounds to approximately one- third that of controls. Steroid/AMS Wounds (Group 6). The most striking difference be- tween these wounds and the control wounds after 1, 2 or 3 days was the almost complete absence of macrophages (Text-figures 2 and 3). At low magnification it appeared as if the level of neutrophils in these wounds was greater than in controls. On quantitation, however, it be- came apparent that the actual area occupied by neutrophils was not increased at 24 or 48 hours but that the absence of macrophages em- phasized the presence of neutrophils, giving an illusory appearance of increased numbers. Although the maximal level of neutrophils was not increased, by 3 days clearance of neutrophils was clearly delayed. In addition, fibrin occupied twice the volume in these wounds as com- pared to controls.

Fibrosis Control Wounds (Groups 1 and 2). Immature fibroblasts were ob- served in control wounds as early as 3 days after wounding, albeit only

60 TExT-FIG 2-His- togram indicating the percentage of wound 50 volume occupied by the various cellular and humoral compo- tE40^ T t vnents (RBCs, collagen, fibrin, neutrophils, fi- broblasts, macro- t30-30 * _ phage/monocytes and :g . _ cell space) 3 days after wounding in groups 1-3 (hatched col- '20- a E_ umns), groups 4 and 5 (striped columns). Counts were per- 10 formed using l-i sections stained Eponwith basic fuchsin and methylene blue. Ver- RBC CG FN i Nt Fi Mac- Snac tical bars indicate SE. *Ko 80 LEIBOVICH AND ROSS American Journal of Pathology 401 ) IMocrophages () Fibrin

30- f

20-

0 t / L..v..4= X ,,,:& ,' 0

40- (§ l\leutrophils Collagen ° 30 ,P TEXT-FIG .3-Com.posite graphs showing the changes in 2 '. / percent wound volume occu- 20- ,^P / pied by various constituents in groups 1-3 (open triangles), o0 ',' groups 4 and 5 (open squares) and group 6 (open circles) 0,,, ,'R,during_$ the course of wound repair. 40- Fibroblasts

30-

10-

01 a 0 2 4 6 8 10 Days in small numbers (Text-figures 2 and 3). Fibroblasts were identified in 1-p sections as elongated, stellate or spindle-shaped cells with round to oval nuclei usually containing one or more prominent nucleoli. By 5 days postwounding the fibroblast was the predominant cell type in the control wounds. At this stage they were large, and their occupied a considerably greater volume than the nucleus. By 7 and 10 days, a marked reduction in the volume of the wounds occupied by fibroblasts occurred. The number of fibroblasts, however was not de- creased. With the decreasing volume occupied by fibroblasts, a marked increase in the area of wound occupied by extracellular material- particularly collagen fibers-was observed. The level of macrophages in control wounds had decreased markedly by 5 days postwounding. In contrast to fibroblasts, macrophages were larger and more irregularly shaped, often with indistinct cyplasmic borders, with characteristically oval or reniform nuclei lacking prominent nucleoli. In 5 and 7 day Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 81 January 1975 wounds, many of the macrophages contained ingested debris and red blood cells. Epithelial over the wound was complete within 5 to 7 days. The advancing tongues of epithelium that migrated from the edges of the wound beneath the scab had generally met by this time forming a sheet of epitheli several cells in depth. AMS Wounds (Group 3). The morphology of 5, 7, and 10 day AMS wounds was identical to that of controls. Steroid Wounds (Groups 4 and 5). As in the control wounds, im- mature fibroblasts were first observed in steroid wounds by day 3 post- wounding. By day 5, fibroblasts were the predominant cell type, and the morphology of the fibroblast at this stage was similar to that of controls. By 7 and 10 days, the volume of wounds occupied by fibro- blasts had markedly decreased, in a manner similar to that of controls. As judged histologically, both from the 1t Epon sections, and from parafn sections stained for collagen, the amount of collagen in the was somewhat lower at 5 days than in controls, but by 7 days, no differences between the steroid and control wounds were observable (Text-figure 3). Fibrin levels at 5 days were more than double those of controls and were still slightly elevated at 7 days. The number of neutrophils present was similar to the controls at all stages. Macrophages were present in these wounds at all stages, but at a considerably reduced level as compared to controls. These cells appeared to be active as judged by the presence of phagocytic vacuoles contain- ing ingested debris, red blood cells and fibrin. It thus appeared that the dose of hydrocortisone used in these ex- periments had no significant effect on either the onset or the rate of proliferation of fibroblasts. Also, little effect on collagen synthesis was observed, as judged both histologically and morphometrically. The principle effect of the steroid appears to be a reduction in the level of macrophages with no effect upon their phagocytic ability. Steroid/AMS Wounds (Group 6). Marked differences in fibrosis be- tween steroid/AMS wounds and control wounds were observed. The steroid/AMS wounds appeared extremely immature in comparison to control wounds in terms of both inflammation and fibroplasia. In marked contrast to the controls, the 5 and 7 day wounds contained many neutro- phils and large amounts of fibrin. Fibroblasts were not observed in the steroid/AMS wounds until 5 days after wounding at which time they were present only in small numbers and were located at the margins of the wounds. Fibroblasts, are, of course, normally the predominant cell type in 5 day wounds, at which time they occupy maximal space, prior to the characteristic reduction in volume by day 7 and 10. The 82 LEIBOVICH AND ROSS American Journal of Pathology low level of fibroblasts in the steroid/AMS wounds by day 10 reflects a marked delay in both the onset and the rate of fibroblast proliferation. The amounts of extracellular debris, fibrin, erythrocytes, serum protein and neutrophils were relatively great, giving the wounds an immature appearance. In addition, reepithelialization of these wounds was mark- edly retarded.

Electron Microscopy of Wound Repair Control and Steroid-Treated Animals (Groups 1-5) Ultrastructural examination of 1, 2 and 3 day wounds confirmed the reduced levels of macrophages in steroid-treated wounds. Macrophages were easily distinguished from fibroblasts-even from the immature fibroblasts present at early stages of wound repair-on the basis of their characteristic fine structure. The macrophages contain a relatively sparse, undilated rough endoplasmic reticulum, often with widely separated groups of attached ribosomes, which appear in en face sections (Figure 2C) as small clusters of two to four ribosomes. Numer- ous free ribosomes are also observed. The Golgi complex in the macro- phages is not as well developed as that observed in the fibroblasts. The macrophage nucleus is generally regular in shape, sometimes ap- pearing reinform or horseshoe shaped and is characterized by a dense uneven layer of chromatin around its circumference. Nucleoli are rarely seen, and when present are generally small. Numerous microvillar pro- jections at the cell periphery are also characteristic. In contrast, the fibroblast has a well-developed rough endoplasmic reticulum with dilated cisternae that ramify throughout the cytoplasm. The attached ribosomes are closely spaced along the RER membrane and in en face views take the form of closely spaced double rows of as many as twenty to thirty ribosomes. Few free ribsomes were observed. In the immature fibroblasts observed at the early stages of wound re- pair, the nucleus occupied a much greater proportion of the total cell volume and numerous cytoplasmic clusters of ribosomes were present, unlike the cells in 5 day wounds, which appeared elongated and had extensive dilated RER and a well-developed Golgi complex, features characteristic of cells actively synthesizing secretory material.2' " The nucleus is markedly different from that of the macrophage, being more irregular in outline, with a characteristically different distribution of chromatin, and one or more prominent nucleoli (Figures 2 and 5). The macrophage generally contains numerous membrane-bounded dense bodies within its cytoplasm. These contain phagocytosed material, Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 83 January 1975 including RBCs, neutrophils, fibrin, -like figures and other un- identifiable debris (Figures 2 and 3). Such inclusions were absent in the fibroblasts although occasional dense bodies were observed. The morphology of the macrophages in steroid-treated wounds was similar to that observed in controls. Formation of peripheral veil-like microvilli was often seen in steroid-treated wounds. However, the functional ability of the macrophages was apparently unaffected, in that phagocytosed material was present within them to an extent com- parable with that of control wounds (Figures 3 and 4A). The morphology of the fibroblasts in steroid-treated wounds was also unaffected, and the cells were surrounded by numerous banded col- lagen fibrils by the fifth day after wounding.

Steroid/AMS-Treated Animals (Group 6) During the first 3 days, electron microscopic examination of steroid/ AMS wounds confirmed the virtual absence of macrophages. Occasional cells that had been tentatively identified as monocytes by light micros- copy were seen to be immature neutrophils rather than monocytes. At the later stages of repair, the immature appearance of the wounds remained confirmed by the presence of considerable neutrophils, erythrocytes, fibrin and serous exudate at days 5, 7 and 10. Those fibroblasts present at the margins of 7 day wounds had relatively normal morphology, although the RER and Golgi were often not developed to the extent observed in control wounds. Collagen fibrils were observed in the extracellular matrix in the vicinity of the fibroblasts (Figure 7). The morphology of the neutrophils was unaffected by either steroids, AMS or combined steroid/AMS treatment. Only the level of neutrophils was altered by steroid/AMS treatment, presumably due to lack of clear- ance of neutrophils from the wounds by macrophages (Text-figure 3 and Figures 6 and 7), and due to the increased numbers of circulating neutrophils in the blood at the later stages of the experiment. Discussion The experiments described in this paper were aimed at elucidating the functions of the macrophage in wound repair by attempting to eliminate this cell from healing wounds and to inhibit phagocytosis by those remaining macrophages. Macrophages at sites of inflammation and wound repair consist of two populations, both probably having their origin in the marrow.20 The first, a minor component, is the "resident" tissue macrophage, which appears to be present in tissues at all times, and under suitable stimuli 84 LEIBOVICH AND ROSS American Journal of Pathology is capable of entering the mitotic cycle and undergoing cell division.21'22 The other, the major component, is directly recruited from hematogenous precursor cells, the monocytes, which themselves are derived from a rapidly dividing pool of cells in the , the promono- cytes.202224 Parabiotic studies have unequivocally demonstrated that most macrophages in sites of inflammation are derived from the mono- cytes in the blood.20

Titration and In Vitro Effects of AMS The effects of AMS on macrophages in vitro are discussed by us in a separate publication.18 AMS in the presence of complement rapidly lysed and killed macrophages, whereas in the absence of complement AMS was not cytotoxic, although phagocytosis was markedly inhibited. When added to macrophages in culture, opsonized erythrocytes nor- mally rapidly adhere to and are phagocytosed by the macrophages. In the presence of AMS (no complement), both adherence and phago- cytosis are inhibited. We have used this effect as the basis of an assay for the titration of AMS. Adherence to and phagocytosis of opsonized erythrocytes by macrophages in culture in the presence of serial dilu- tions of AMS was directly observed. The experiments described in this paper showed that local sub- cutaneous administration of AMS had no observable effect on the macro- phages in healing wounds, since both the number and activity of the macrophages was unaffected. Although an initial 40% drop in the num- ber of circulating monocytes was induced by either AMS or NRS in- jections, this was not maintained in either case, and by day 3 essentially normal blood monocyte levels were observed which were maintained throughout the course of the experiment. Feldman et al,25 studying the specificity of AMS, showed that AMS reacted with a surface present in tissue macrophages, such as peritoneal, splenic and alveolar macrophages, but not in their precursor cells in the blood and bone marrow, the monocytes and , respectively. This observa- tion, together with that of Virolainen et ,26 who demonstrated that bone marrow cells and monocytes maintained in culture for several days developed a macrophage-specific antigen that was not previously detectable, go far toward explaining the apparent inability of AMS to induce a monocytopenia in the experiments described in this paper and to be effective in vivo in previous studies by other investigators.11"2' 2543 Also, indirect evidence from our laboratory 18 suggests that macro- phages affected by AMS in vitro release substances chemotactic for other unaffected macrophages, which rapidly adhere to and phagocytose Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 85 January 1975 the affected cells. In vivo, AMS appears to react only with mature macrophages. These affected cells are then rapidly removed and re- placed by newly synthesized, hematogenously borne precursors which do not react with the antibody. Any effects of the AMS are thus rapidly masked.

In Vivo Effect of Hydmctisone As AMS alone had no effect on either the number or the phagocytic activity of macrophages in healing wounds under conditions where rapid replacement of affected cells by hematogenous precursors was possible, it became necessary to devise a means of eliminating the source of replacement cells. Recently, Thomson and Van Furth showed that administration of hydrocortisone to mice resulted in a rapid and marked decrease in the level of circulating monocytes.9'10 The dura- tion of the monocytopenia was dependent on the nature and dose of the administered steroid. Reduction of mononuclear at sites of inflammation, a characteristic feature of steroid treatment,"49 can be largely accounted for on the basis of this reduction in blood monocytes. Thomson and Van Furth" 10 demonstrated that the steroid treatment resulted in rapid sequestration of monocytes in some as yet unidentified depot, rather than to a direct lytic effect on the mono- cytes. The numbers of macrophages present in the peritoneal cavity were not affected by this treatment. Recruitment of monocytes to a site of inflammation was, however, markedly decreased due to the monocytopenia. We utilized this ability of hydrocortisone (admin- istered as a subcutaneous insoluble depot to give slow, sustained release of steroid over an extended period) to induce a monocytopenia in guinea pigs and thus to eliminate the principle source of the macro- phages in the healing wouds. Our results demonstrate that hydro- cortisone alone reduces the numbers of macrophages in wounds to approximately one third. During steroid treatment, those macrophages observed in the wounds appeared to be normal in terms of both morphology and phagocytic ability. It is not clear, however, whether the macrophages in these wounds originate from immigration of mono- cytes mobilized from an unidentified depot as the result of the injurious stimulus, or whether they result from local proliferation close to the site of wounding. This reduction in macrophage levels was accompanied by some inhibition of wound debridement, as evi- denced by an increased level of fibrin and a reduced rate of disappear- ance of both fibrin and neutrophils. Previous studies of the effects of adrenal steroids upon 86 LEIBOVICH AND ROSS American Journal of Pathology are both conflicting and confusing.5069 It is particularly difficult to evaluate the significance of many of these investigations, as it is well known that different animals differ in their susceptibility to these agents. The rabbit in particular exhibits a marked susceptibility, and many of the studies of the effects of steroids on wound repair have been done using this animal.5051'57'60'61'67-69 The rat also exhibits greater susceptibility than the guinea pig5 and has also been the animal of choice in a number of investigations.52'546'58,59,62-66 Clearly, under the dosage regime which we used in our experiments with guinea pigs, the course of wound repair was not significantly altered, either in terms of the onset or the degree of fibrosis. In our experiments, the numbers of fibroblasts in the wounds of the steroid-treated animals were the same at each stage of repair as in the controls. Their mor- phology was unaffected, since no change in RER, Golgi complexes or other subcellular constituents were observed. Surprisingly, as judged by morphometric analysis, collagen synthesis appeared to be unimpaired in these wounds when compared with controls. When viewed in the electron microscope, collagen fibrils with characteristic banding were present in the extracellular matrix. An effect of hydrocortisone on the remodeling phase of wound repair however, cannot be ruled out by these experiments. Measurements of tensile strength of hydro- cortisone-treated wounds have indicated that the effect of hydro- cortisone may be primarily on this later, remodelling stage of repair.64'6 Since it is impossible to judge the status of collagen synthesis by mor- phologic or morphometric analysis alone, further biochemical studies will be required before the significance of this observation can be ascertained.

In Vivo Effect of Antimacrophage Serum Plus Hydrocortisone While local administration of AMS alone had no observable effect on the macrophages, when used in conjunction with hydorcortisone (steroid/AMS wounds) it resulted in the virtually complete dis- appearance of macrophages from the wounds. It seems that, under conditions of a monocytopenia, where monocyte immigration is reduced to a minimum, AMS is capable of reacting with and destroying most of the residual macrophages in the wounds. In such macrophage- deficient wounds, clear differences in debridement and fibrosis were observed. Wound fibrin levels were greatly elevated over controls, and were significantly higher than those observed in steroid-treated wounds (steroid alone), where macrophage levels were reduced to one-third that of control wounds. While the absolute level of wound Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 87 January 1975 neutrophils was not altered in the first 2 days, clearance of neutrophils from the wounds was markedly inhibited, resulting in inordinately high neutrophil levels during the later stages of repair. Seven day wounds exhibited remarkable immaturity in terms of their histologic appearance and contained considerable neutrophils, fibrin, erythrocytes and serous exudate. Relatively few fibroblasts were present, and were found mainly at the wound margins. Where fibroblasts were observed, however, collagen synthesis appeared to be unimpaired, as judged histologically and with the electron microscope. The fibroblasts ap- peared to be less mature in terms of the development of both their RER and Golgi complexes. As Text-figure 3 shows most clearly, both the onset and the rate of proliferation of fibroblasts in the steroid-AMS wounds was slower than that in control wounds, reflecting the immature appearance of these wounds at the later stages of repair.

Role of Maophage in Wound Repair The primary effects of the macrophage depletion in steroid/AMS wounds were: 1. A severe lack of wound debridement. This clearly implicates the macrophage as the principal phagocytic cell in wound repair. While the early arrival of large numbers of neutrophils in wounds provides an effective local barrier against bacterial invasion,4'70 no other role for this prominent early component in the inflammatory response was found in previous studies of wound repair. The studies described in this paper clearly demonstrate that, in contrast to the neutrophil, the macrophage is essential for wound debridement, since severe inhibition of debridement resulted from absence of these cells in the wounds. 2. A marked delay in fibrosis, in terms of both the onset and the rate of proliferation of fibroblasts. This delay in fibrosis may be due to a variety of causes which cannot be conclusively distinguished on the basis of these in vivo experiments. Firstly, an essential prerequisite to fibroblast proliferation might be adequate wound debridement. It could be that the presence of a fibrin meshwork containing cells and other miscellaneous debris acts as a physical barrier to the immigration into the wounds and the subsequent proliferation of fibroblasts. How- ever, the results for the wounds treated with steroid alone (where fibrosis was virtually unaffected, while debridement was somewhat impaired-although not to the same extent as in steroid/AMS wounds) tend to argue against this hypothesis as the only factor involved. Also, previous investigators demonstrated that inhibitors of fibrinolysis 88 LEIBOVICH AND ROSS American Journal of Pathology served to promote rather than to impair fibroblast proliferation in subcutaneously implanted plasma clots.71 Alternatively, it is impossible that inhibitors of fibroblast prolifera- tion are present in the wounds and that macrophages are required to break down or inactivate these inhibitors, thus permitting cell proliferation. It is equally possible that macrophages are required for the production of a factor (or factors) that promote fibroblast pro- liferation. Such factors could be produced directly by or as a result of activation of macrophages. On the other hand, such factors might be indirectly produced as the result of breakdown of specific wound components by the macrophages. We are currently investigating these possibilities. Note Added in Proof The method is based on earlier observations by Knudson et al (Knudson SM, Schwarz MR, Perkins WD: Phagocytosis of ALS treated lymphocytes: prevention of antimacro- phage serum. Proceedings of the Sixth Leucocyte Culture Conference. Edited by MR Schwarz. New York, Academic Press, Inc, 1972, pp 411-421). References 1. Ross R, Benditt EP: Wound healing and collagen formation. I. Sequential changes in components of guinea pig skin wounds observed in the electron microscope. J Biophys Biochem Cytol 11:677-700, 1961 2. Ross R: The fibroblast and wound repair. Biol Rev (Camb) 43:51-96, 1968 3. Gillman T: On some aspects of collagen formation in localized repair and in diffuse fibrotic reactions to injury, Treatise on Collagen. Vol. 1. Edited by BS Gould. New York, Academic Press, Inc, 1968, pp 331-407 4. Simpson DM, Ross R: The neutrophilic leukocyte in wound repair: a study with antineutrophil serum. J Clin Invest 51:2009-2023 5. Simpson DM, Ross R: Effects of heterologous antineutrophil serum in guinea pigs: hematologic and ultrastructural observations. Am J Pathol 65: 49-102, 1971 6. Page AR, Good RA: Studies on cyclic neutropenia. Am j Dis Child 94: 623-661, 1957 7. Page AR, Good RA: A clinical and experimental study of the function of neutrophils in the inflammatory response. Am J Pathol 34:645-669, 1958 8. Ward PA, Hill JH: C5 chemotactic fragments produced by an in lysosomal granules of neutrophils. J Immunol 104:535:543, 1970 9. Thomson J, Van Furth R: The effect of glucocorticosteroids on the kinetics of mononuclear phagocytes. J Exp Med 131:439-442, 1970 10. Thompson J, Van Furth R: The effect of glucocorticosteroids on the pro- liferation and kinetics of promonocytes and monocytes of the bone marrow. J Exp Med 137:10-21, 1971 11. Marsman AWJ, van der Hart M, Loghem JT: Antigenic differences between macrophages and lymphocytes. Clin Exp Immunol 6:899-903, 1970 12. Unanue E: Properties of some use of antimacrophage antibodies. Nature rLond] 218:36-38, 1968 13. Henney CS: Quantitation of the cell mediated . I. The number of cytolytically active mouse lymphoid cells induced by immunization with allogeneic mastocytoma cells. j Immunol 107:1558-1566, 1971 Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 89 January 1975

14. Humphrey JH: The mechanism of Arthus reactions. II. The role of poly- morphonuclear leukocytes and in reversed passive reactions in the guinea-pig. Br J Exp Pathol 36:283-289, 1955 15. Karovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electro microscopy. J Cell Biol 27:137A-138A, 1965 16. Huber JD, Parker F, Odiand GF: A basic fuchsin and alkalinized methelene- blue rapid stain for epoxy-embedded tissue. Stain Technol 43:83-87, 1968 17. Weibel ER, Kister GS, Scherl WF: Practical stereological iethods for morphometric cytology. J Cell Biol 30:23-438, 1966 18. Leibovich SJ, Ross R: Macrophages and antimacrophage serum, Mono- nuclear Phagocytes. Edited by R Van Furth. Philadelphia, FR Davis Co (In press) 19. Greenlee TK, Ross R: The development of the rat flexor digital : a fine structure study. J Ultrastt Res 18:354-376, 1967 20. Volkman A, Gowans JL: The origin of macrophages from bone marrow in the rat: Br J Exp Pathol 46:62-70, 1965 21. Ryan GB, Spector WG: Macrophage turover in inflamed . Proc R Soc Lond (Biol) 174:269-292, 1970 22. Volkmnan A: A current perspective in . Curr Top Pathol 54:76-94, 1971 23. Van Furth R, Cohn ZA: The origin and kinetics of mononuclear phagocytes. J Exp Med 128:415-43, 1968 24. Virolainen M: Hematopoietic origin of macrophages as studied by chromo- some markers in mice. J Exp Med 127:943-951, 1968 25. Feldman JD, Tubergen DG, Pollock EM, Unanue ER: Distribution of a macrophage specific antigen. Cell Immunol 5:325-337, 1972 26. Virolainen M, Lahti A, Hayri P: Advances in Experimental Medicine and Biology. Vol 15. Edited by NR DiLuzio, K Flemng. Proceedings of the Ludwig Ashoff Memori Meeting of the Reticuloendothelial Society, Freiberg, Germany, August 1970. New York, Plenum Press, 1971 27. Cayeux P, Panijel J, Cluzan R, Leviain R: Streptococcal arthritis and cardiopathy epermentay induced in white mice. Natue [Lond] 212:608- 691, 1966 28. Panijel J, Cayeux P: Immunosuppressive effects of macrophage antiserum. 14:769-780, 1968 29. Jennings JF, Hughes LA: Inhibition of phagocytosis by anti-macrophage antibodies. Nature [Lond] 221:79-80,1969 30. Despnt JP, Cruchaud A: In vivo and in vitro effects of anti-macrophage serm. Nature [Lond] 223:838-839, 1969 31. Hirsch MS, Gary GW Jr, Murphy FA: In vitro and in vivo properties of antimacrophage sera. J Immunol 102:656-661, 1969 32. Loewi G, Temple AP, Axelrad M: A study of the effects of antimacrophage sera. Immunology 16:99-106, 1969 33. Argyris BF, Plotin DH: Effects of antimacrophage serum on antibody production and phagocytosis in mice. J Immunol 103:372-373, 1969 34. Jehn UW, Musher DM, Weinstein L: The effect of antimacrophage serum on macrophage-lymphocyte interaction in vitro. Proc Soc Exp Biol Med 134:241-243, 1970 35. Ziff, M, Hurd ER, ILmmel, Jasin HE:: The effect of cytostatic agents on the kinetics of mononuclear phagocytes.18 pp 282297 90 LEIBOVICH AND ROSS American Journal of Pathology

36. Ptak W, Porwitt-Bobr Z, Chlap Z: Transformation of hamster macrophages into giant cells with antimacrophage serum. Nature [Lond] 225:655-657, 1970 37. Feldman JD, Unanue ER: Role of macrophages in delayed hypersensitivity. II. Effects of antimacrophage antibody. Cell Immunol 2:275:282, 1971 38. Shortman K, Palmer J: The requirement for macrophages in the in vitro immune response. Cell Immunol 2:399-410, 1971 39. Jason HE, Lennard D, Ziff M: Studies on antimacrophage globulin. Clin Exp Immunol 8:801-814, 1971 40. Gaillily R: In vitro and in vivo studies of the properties and effects of anti- macrophage sera (AMS). Clin Exp Immunol 9:381-391, 1971 41. Sharma K, Grone M, Sampson D, Guinan P, Murphy GP: Immuno- suppressive effects of antimacrophage serum and antithymocyte serum on mouse skin allografts. J Med 3:199-211, 1972 42. Ptak W, Cichoki T: The mechanisms of antimacrophage serum-induced fusion of hamster macrophages. Acta Histochem 44:116-121, 1972 43. Schroit AJ, Geiger B, Gallily R: The capacity of macrophage components to inhibit antimacrophage serum activity. Eur J Immunol 3:354-359, 1973 44. Dougherty TF, Sehneebeli G: Role of cortisone in regulation of inflamma- tion. Proc Soc Exp Biol Med 75:854-859, 1950 45. Cummings MM, Drummond MC, Michael M Jr, Bloom WL: The influence of cortisone on artificially induced peritoneal exudates. Bull Johns Hopkins Hosp 90:185-191, 1952 46. Lurie MB, Zappasodi P, Dannenberg AM, Cardona-Lynch E: The effect of cortisone and A.C.T.H. on the pathogenesis of . Ann NY Acad Sci 56:779-792, 1953 47. Gell PGH, Hinde IT: The effect of cortisone on macrophage activity in mice. Br J Exp Pathol 34:273-275, 1953 48. Craddock CG, Winkelstein A, Matsuyuki, Lawrence JS: The immune re- response to foreign red blood cells and the participation of short lived lympho- cytes. J Exp Med 125:1149-1172, 1967 49. Nicol T, Quantock DC, Vernon-Roberts B: The effects of steroid hormones on local and general reticulo-endothelial activity: relation of steroid structure to function, The Reticuloendothelial System and . Edited by NR DiLuzio, R Paolette. New York, Plenum Press, 1967, p 221 50. Ragan C, Howes EL, Plotz CM, Meyer K, Blunt JW: Effect of cortisone on production of in the rabbit. Proc Soc Exp Biol Med 72:718-721, 1949 51. Bangham AD: The effect of cortisone on wound healing. Br J Exp Pathol 32:77-84, 1951 52. Baker BL, Whittaker WL: Interference with wound healing by local action of adrenocortical steroids. Endocrinology 46:544-551, 1950 53. Creditor MC, Bevans M, Mundy WL, Ragan C: Effect of ACTH on wound healing in humans. Proc Soc Exp Biol Med 74:245-247, 1950 54. Alrich EM, Carter JP, Lehman EP: The effect of ACTH and cortisone on wound healing: an experimental study. Ann Surg 133:783-789, 1951 55. Meadows EC, Prudden JF: A study of the influence of adrenal steroids on the strength of healing wounds. 3:841-848, 1953 56. Savlov ED, Dunphy JE: The healing of the disrupted and resutured wound. Surgery 36:363-370, 1954 Vol. 78, No. 1 MACROPHAGES AND WOUND REPAIR 91 January 1975

57. Bukhmonova AI: Histofunctional changes in a wound at different concen- trations of the hormones of the adreal and thyroid glands. Proc Acad Sci USSR 134:1256 1259,1960 58. Pearce CW, Foot CN, Jordan GL Jr, Law SW, Wantz GE: The effect and interrelation of , cortisne, and protein nutrition on wound healing. Surg Gynecol Obstet 111:274-284, 1960 59. Calwell FT Jr, Dnohue P, Rosenberg B: Rate of gain of tensike strength of abdominal woxmds in rats. JAMA 179:773-775, 1962 60. Davidson EA, Small W: Metabolism in vivo of conective-tissue mucopolyr- saccharides. I. Cbondroitin sulate C and keratosulfate of nucleus pulpous. Biochim Biophys Acta 69:445-452, 1963 61. KCaplan D, Fisher B: Tbe effect of methylprednisolone oKn mucopoly- saccharides of rabbit vitreou huimn and costal cartilage. Biochim Biophys Acta 83:102:112, 1964 62. DiPasquale G, Steinetz BG: Relationship of food intake to the effect of cortisone acetate on slidn wound heaing. Proc Soc E:xp Biol Med 117:118:121, 1964 63. Sandberg N: Time relationship between adinistration of cortisoe and wond healing in rats. Acta Chir Scan}d 127:4-455, 1964 64. Mildconon IL, Lampiaho K, Kulonen E: Effect of thyroid hormones, somato- trophin, insuln and corticosteroids on synthesis of collagen in granukition tissue both in VIVO and in vitro. Acta Endociol 51:23-31, 1966 65. DiPasquale G, Tripp L, Steinetz BG: Effect of locaHy applied anti-inflam- matory substances on rat skidn wounds. Proc Soc Exp Biol Med 124:404-47, 19ff7 66. DiPasquale D. Tnp LV, Steinetz BG: Effect of Iysomal labihizers and proinflammaw s%ubtances on connetve fisse repair as masured by tensile stre gth. Proc Soc Exp Biol Med 127:529-M2, 1968 67. Ebhrlich HP, Hunt TKZ: Effect of cortisone and vitamin A on wound healing. Ann Surg 1ff7:324-328, 1968 68. Hunt TK, Ehrlich BP, Garcia JA, Dunphy JE: Effect of vitamin A on revers- ing the inhibitory effect of cotie on healing of open wounds in animals and man. Ann Surg 170:633-641, 1969 69. Joseph J, Tydd M: Thne effects of cortisone acetate on tisse regeneration in the rabbit's ear. J Anat 115:445-460, 1973 70. Steigbigel RT, Lambert LH Jr, Remington JS: Phagocytic and bactericidal poetes of normal human mowoye. J Clin Invest 53:131-142, 1974 71. Kwaan HC, Astrup T: Tise repair in presen}ce of locally applied inhibitors of fibrinolysis. Exp Mol Pathol 11:82488, 1969 Acknowledgments lbe autors gratefully anwege the exceDet technical asssance of Ms. Lynne Phil+, Ms. Mary Stewart, Ms. Willie Mae Yg-o and Mr. William Fulford in these studies. Thxey are tlD Mr. Johsel Nakung fo the preparation of the mi-crograph plates. D.Lord i-s a postdoctoral fellow of the Atritis Fonation, and grateul a recedeipt of travel funids from the Wellcome Foundation; his present add is Deatment of Biological Ulrsucture, Weizmann Inttute of Science, Rehovot, Israel. 92 LEIBOVICH AND ROSS American Journal of Pathology

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Fig 1-Electronmicrograph of a 3 day control (untreated) guinea pig skin wound. A macrophage (M), with typical dense bodies (db) and phagocytic vacuoles (va) is apparent, as are a number of immature fibroblasts (F). The macrophage can be differentiated from the fibroblasts by its relative paucity of rough endoplasmic reticulum (rer), the different distribution of its nuclear chromatin and by its lack of prominent nucleoli. In contrast, the fibroblast, even at this early stage of matu- ration, contains extended cisternae of rough endoplasmic reticulum, and large prominent nucleoli (x 5300). Fig 2A-A typical macrophage from a 3 day control wound (treated with normal rabbit serum alone). Numerous dense bodies (db) and vacuoles (va) are present. The rough endoplasmic reticulum (rer) is sparse, and numerous free ribosomes are present. Peripheral microvilli, a characteristic feature of macrophages, can be seen (X 4600). B-A normal section through a segment of macrophage show- ing segments of the Golgi vesicles (G) and several parallel cisternae of rough endoplasmic reticulum. The random distribution of ribosomes attached to the membranes and the rather lengthy segments of membrane lacking attached ribosomes are characteristic of rough endoplasmic reticulum of macrophages (X 17,000). C-An en face section of an area of rough endoplasmic reticulum of a macrophage, in which the distribution of attached ribosomes can be seen. The ribosomes attached to the membranes of the rough endoplasmic reticulum consist of small aggregates and have a strikingly different appearance in their distribution when compared with those in the fibroblast (X 17,000). , . 1t _ .... ., A. 4% . .i '*.1. -. ..I _&.t ' .' IL

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Fig 3-This micrograph demonstrates an immature fibroblast (F) next to a mono- nuclear (M) in a 3 day wound from a guinea pig treated with gluco- corticosteroid alone. The extensive rough endoplasmic reticulum (rer) in the fibroblast can be seen, while in the macrophage it is not well developed. Numerous free ribosomes are present in the macrophage. The characteristic large nucleolus in the fibroblast helps to differentiate it from the mononuclear phagocyte, which has a much smaller, less prominent nucleolus. The periphery of the macrophage contains numerous fronds or microvillous-like structures. The glucocorticosteroid treatment has not (x8000). affected the typical morphology of the cells in the wound 4,~~~~~~~~~~~~~~47

Fig 4A-An electronmicrograph of a 3 day skin wound from a glucocorticosteroid treated guinea pig. Two neutrophilic leukocytes (N) which have been ingested by a macrophage (M) can be seen in various stages of degeneration. The macrophage is surrounded by numerous intact neutrophils. The phagocytic ability of the macrophage appears to have been unaffected by the glucocorticosteroid treatment (x 4000). B-An electronmicro- graph from the central region of a 3 day skin wound from a guinea pig treated with gluco- corticosteroid and antimacrophage serum (steroid/AMS animals). A number of neutrophils are present, and clear evidence of phagocytosis, such as the large phagocytic in the cell at the lower left of the micrograph (arrow), can be seen. ConsideLrable fibrin is present in the extracellular space, and numerous erythrocytes are also present. No macro- phages can be seen (x 4000). --- .: ... It P-. I-f..v Z

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Fig 5-Electronmicrograph from the area at the center of a 7 day control (NRS- treated) guinea pig skin wound. A typical macrophage (M) can be seen together with a number of fibroblasts (F). The cell in the lower central region is sectioned so that its nucleus is not apparent The extensive rough endoplasmic reticulum (rer) of the cell is, however, clearly visible. Numerous fine collagen fibrils (coll) can be seen in the extracellular spaces surrounding the fibroblasts (X 4000). Ir f

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Fig 6-Electronmicrograph of the area at the center of a 7 day wound from a glucocorticosteroid and antimacrophage serum (steroid/AMS)-treated guinea pig. In contrast to the control wound shown in Figure 5, numerous neutrophils are present, a feature not normally observed in control wounds at this late stage of wound healing, neutrophils being virtually completely absent at this time. (X 5500). - 4~~~~~~~~~~~~~~~~~~~O

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Fig 7-Ekectronmicrograph of an area close to the margin of a 7 day wound from a guinea pig treated with glucocorticosteroid and antimacrophage serum (steroid/AMS animal). Fibroblasts (F) and neutrophils (N) are present, showing the abundance of neutrophils in the wounds of this group of animals at this late stage of wound healing. The fibroblasts appear to be normal in terms of the development of their rough endoplasmic reticulum (rer) and Golgi complex (G) (X 5500). LEIBOVICH AND ROSS American Journal MACROPHAGES AND WOUND REPAIR of Pathology

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