JOURNAL OF CELLULAR PHYSIOLOGY 148:157-173 (1991)

Direct Evidence for Spatial and Temporal Regulation of Transforming Growth Factor PI Expression During Cutaneous Wound Healing CYNTHIA J.M.KANE,* PATRICIA A. HEBDA, JONATHAN N. MANSBRIDGE, AND PHILIP C. HANAWALT Departments of Biological Sciences (C.I.M.K., P.C.H.) and Dermatology (C.I.M.K.,I.N.M.), Stanford University, Stanford, California 94305; Department of Dermatology (P.A.H.1, University of Pittsburgh, Pittsburgh, Pennsylvania J526 1 The expression of transforming growth factor (TGFP1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGFPl constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGFPl present in normal skin as previously shown. TGFPl may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGFPl protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness porcine skin. Anti-CC reactive TGFPl in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGFPl protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGFPl activity. One day post-injury anti-CC reactive TGFPl is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGFPl is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGFPl from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGFPl activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGFPl disappears while constitutive anti-LC reactiveTGFP1 persists. Thus, changes in the structureor conformation of TGFPl , its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.

Five distinct tranasforming growth factor type p (TGFp) genes, designated TGFPl-5, have been identified (Derynck et al., 1985,1988; de Martin et al., 1987; ten Dijke et al., 1988; Jakowlew et al., 1988; Kondaiah et al., 1990). TGFP1-3 have been isolated from humans (Derynck et al., 1985, 1988; de Martin et al., 1987; ten Dijke et al., 1988). Each gene encodes a distinct peptide which dimerizes to form an inactive TGFp precursor protein (Derynck et al., 1985). The latent protein is glycosylated, phosphorylated, secreted, and cleaved to yield a biolo ically active 25-kD homodimeric peptide as modeled y L ons and Moses (1990). The significance of multiple gighly conserved TGFp genes has not

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been established. The biochemistry and biological activity of TGFPl has been the best characterized of the TGFP peptides. TGFPl is both a growth factor and a growth inhibitor. TGFPl dramatically modulates the proliferation and activity of diverse mammalian cells. It alters the response of most cells to multiple external stimuli in vitro including mitogens, differentiating agents, and extracellular matrix. (See Roberts and Received September 7, 1990; accepted March 12, 1991. *:To whom reprint requestskorrespondence should be addressed at: Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.

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Sporn, 1990; Barnard et al., 1990; Lyons and Moses, 1990; for recent reviews.) The principal function of TGFpl may be to coordinate complex multicellular physiological events normally associated with growth and development. The normal cutaneous response to injury is a highly ordered and well-coordinated process involving both epidermal and dermal components. A role for TGFpl in regulation of cellular proliferation, differentiation, and chemotaxis during cutaneous wound healing is suggested. Previous studies by Kane et al. (1990)have shown that TGFPl is expressed in normal human adult skin and that the pattern of expression is altered in psoriasis which exhibits aspects of the differentiation phenotype typically associated with cutaneous wound healing (Mansbridge and Knapp, 1987). Cells resident in the epidermis and the dermis synthesize, secrete, and respond to TGFpl in vitro including keratinocytes, fibroblasts, endothelial cells, Langerhans cells, lymphocytes, macrophages, and monocytes (Roberts and Sporn, 1990; Barnard et al., 1990; Lyons and Moses, 1990). Proliferation of epidermal keratinocytes in culture is reversibly inhibited in the late G1 phase by TGFPl at picomolar concentrations (Shipley et al., 1986; Pietenpol et al., 1990; Laiho et al., 1990). Initiation of migration of keratinocytes in explant cultures is stimulated by TGFPl at similar concentrations (Hebda, 1988).TGFPl modulates the differentiation phenotype of keratinocytes as previously reported by Mansbridge and Hanawalt (1988). Cultured keratinocytes are induced by TGFpl to express keratins 6 and 16, differentiation markers associated with epidermal regeneration and hyperproliferation, while expression of the normal epidermal differentiation marker keratin 1 is suppressed. Proliferation of fibroblasts can be stimulated, inhibited, or not affected by TGFPl in vitro depending on the culture conditions and the cell growth and differentiation state (reviewed by Roberts and Sporn, 1990). When subcutaneously injected in newborn mouse skin (Roberts et al., 1986) or introduced into wound chambers in rats (Sporn et al., 1983) it promotes synthesis and accumulation of granulation tissue by fibroblasts and stimulates angiogenesis, both essential events in cutaneous wound repair. By analogy to in vitro results, TGFPl can selectively control the inflammatory response which occurs following injury. TGFpl is secreted by platelets, lymphocytes, monocytes, and activated macrophages. TGFPl stimulates the beneficial anabolic activity of macrophage-derived growth factors while inhibiting the destructive res iratory burst of activated macrophages, and inhibits t e proliferation and activity of lymphocytes, neutrophils, and eosinophils (reviewed by Wahl et al., 1989). In addition to control of proliferation and differentiation, directed cellular migration is central to wound repair. TGFpl is chemotactic for keratinocytes, fibroblasts, inflammatory cells, monocytes, macrophages, and endothelial cells (reviewed by Roberts and Sporn, 1990; Lyons and Moses, 1990; Wahl et al., 1989). Thus, by analogy to in vitro observations, an increase in TGFpl activity might be expected to correlate with the switch from a normal to a regenerative phenotype, characteristic of wound healing, following cutaneous injury. TGFp activity has been detected in percutaneous wound chamber fluid and the level varies in a

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temporal manner throughout healing (Cromack et al., 1987). Topical application of TGFPl to cutaneous wounds in orcine and rat skin promotes healing (Mustoe et a,!. 1987; Hebda, 1989; Pierce et al., 1989). However, it has been impossible to determine the specificity and the mechanisms of TGFPl activity during cutaneous wound healing because its role in the normal healing pathway is unknown and can only be hypothesized from in vitro studies. We have previously shown by immunohistochemistry the expression and localization of TGFPl protein in normal human skin (Kane et al., 1990). Analysis of this expression in situ upon cutaneous in'ury and at various phases of the repair process wou d provide a rational basis for evaluation of TGFpl activity. Changes in the expression or localization of TGFPl relative to uninjured skin could establish a role for TGFPl in normal cutaneous injury response. Using immunohistochemistry this study documents changes in the expression of TGFPl and its relocalization in normal adult human and porcine skin upon injury. The results indicate that the structure or conformation, and perhaps the activity, of TGFpl is spatially and temporally modulated during normal wound healing. Correlation of these changes in TGFPl with the physiology of the healing process indicates a putative role for TGFPl in regulation of the complex array of cellular activity occurring during cutaneous repair. MATERIALS AND METHODS Antibodies The two TGFp1-s ecific rabbit polyclonal antibodies have been previous y described and characterized (Ellingsworth et al., 1986; Flanders et al., 1988). The antibodies were prepared to different synthetic preparations of a peptide corresponding to the first 30 amino acids of the amino-terminus of mature TGFp1. Anti-CC antibody and the similarly prepared nonimmune rabbit IgG control were the generous gifts of L. Ellingsworth, Collagen Corporation, Palo Alto, CA. Anti-LC antibody and the corresponding control nonimmune rabbit IgG were kindly provided by K. Flanders, National Cancer Institute, NIH, Bethesda, MD. The anti-CC (Ellingsworth et al., 1986) and anti-LC (Flanders et al., 1988) antibodies have been extensively characterized with regard to TGFpl specificity in skin (Kane et al., 1990) by 1) omission of primary antibody, 2) substitution of nonimmune rabbit IgG, pre ared in the same manner as the immune IgG, for t e same concentration of primary antibody, 3) depletion of TGFPl-specific binding by preincubation of anti-CC antibody with TGFP1coupled Sepharose (Heine et al., 19871, and 4)blocking of anti-LC antibody binding by preincubation with a 20-fold molar excess of the LC peptide for 2 hours at room temperature prior to application as primary antibody (Flanders et al., 1989). The affinity of anti-CC and anti-LC for distinct epitopes of TGFPl has been established (Heine et al., 1987; Flanders et al., 1989). Anti-LC has a high affinity for synthetic peptides corresponding to amino acids 1-10 and 21-30 of the amino-terminus of mature TGFP1. Anti-CC has a lower affinity for amino acids 1-10 and appears to bind a discontinuous epitope not presented by the short decameric peptides. The TGFPl specificity of anti-CC and anti-LC have been documented by western blots (El-

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lingsworth et al., 19861, radioimmunoassay, and enzyme-linked immunosorbent assay analysis (Flanders et al., 1989) and demonstrated by immunohistochemistry in skin (Kane et al., 1990). Human suction blister injury Normal human skin specimens were obtained from seven human volunteers by 3 mm punch biopsy under xylocaine local anesthesia. Suction blisters were preared on the inner surface of the upper arm of a single uman volunteer as previously described (Mansbridge and Knapp, 1987) by the method of Kiistala and Mustakallio (1967). The blister roofs, detached from underlying tissue at the basement membrane, were removed immediately and the wound was left open to air. Single punch biopsies of 4 mm were removed at 24 hours and 3 and 7 days after injury. Biopsies were immediately fixed in Bouin’s solution and processed for immunohistochemistry . Human incisional injury Human adult facial skin removed at myeloplasty was placed in buffered saline and processed within an hour of surgery. Large specimens were cut into approximately 5 mm squares and placed in phosphate-buffered saline alone or supplemented with either 0.3 mgiml leupeptin or 1 mM ethylenediaminetetraacetic acid (EDTA) for 5 minutes. A scalpel incision was made through the epidermis into the dermis near the center of the specimen. The response to injury was stopped at various time points following incision by fixation in 10% neutral-buffered formalin. Specimens were processed in Bouin’s solution and prepared for immunohistochemistry . Porcine dermatome excision injury Shallow partial thickness wounds were made in porcine skin as previously described (Eaglstein and Mertz, 1978; Mertz et al., 1986). Five white female Yorkshire pigs (20-30 pounds, approximately 2-3 months old) of similar stock from a single supplier were housed in individual pens in an AAALAC accredited animal facility with controlled temperature (19-21OC) and light (12 hr:12 hr, 1ight:dark). The pigs were fed a basal diet ad libitum. The hair was clipped with standard animal clip ers and the skin on the back and sides was pre ared or wounding by washing with a mild soap an water and rinsing. Other antiseptics were not used because of the potential effect on the healing process. Before surgery, the pi s were anesthetized with ketamine (300 mg i.m.) anI f halothane (3%, open mask) administered with nitrous oxide and oxygen (40:60,5 Limin). On each animal small rectangular wounds, approximately 1 cm2 and 0.3 mm deep, were made in the paravertebral and thoracic areas with a Castrovie‘o electrodermatome (Storz Instruments, St. Louis, MA). This standard wound has been shown to achieve the com lete removal of epidermis and the most superficial! ( ermis, leaving the epidermal appendages intact (Eaglstein and Mertz, 1978; Winter, 1962). Punch biopsies of 6 mm were removed at 0.5,1,2,8,24, 96 hours and 7, 10, and 12 days post-injury and included wound area and adjacent skin tissue. Biopsies were taken in duplicate from individual animals in all

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cases and each time point was taken from at least two animals. Biopsies were immediately fixed in Bouin’s solution and processed for immunohistochemistry. All four specimens for each timepoint presented similar immunohistochemical results relative to the stage of individual healing. Tissue fixation and immunohistochemical staining Biopsies were fixed at 4°C in Bouin’s solution [saturated a ueous picric acid:formalin:acetic acid (15:5:1)1 overnig t. Tissue was dehydrated, embedded in paraffin, and sectioned at 4-6 pm. Sections were deparaffinized, rehydrated, endogenous peroxidase blocked by 30 minute incubation in 1%hydrogen peroxide in methanol, and permeabilized by treatment with 1 mgiml hyaluronidase, type I-S (Sigma, St. Louis, MO), in 0.1 M sodium acetate, 0.15 M sodium chloride, pH 5.5, for 30 minutes at 37°C. Nonspecific protein binding was blocked with 5% calf serum in phosphate-buffered saline. Anti-CC antibody and its control I G were incubated with sections at 12.5 pgiml for 2 ours at room temperature. Anti-LC antibody and its control IgG were incubated with sections at 10 p,giml for 2 hours at room temperature. Unbound antibody was removed by washing with 5% calf serum in phos hatebuffered saline. Bound antibody was localizef with biotinylated goat anti-rabbit IgG, an avidin-peroxidase Vectastain kit (Vector Laboratories, Burlingame, CA), and 3,3’-diaminobenzidine (Polysciences, Warrington, PA) with hydrogen peroxide as peroxidase substrate. Sections were counterstained with Gill’s hematoxylin (Sigma, St. Louis, MO). Sections were photographed with an Olympus BH-2 microscope using ND12 and 0.5CT filters on Kodak Ektachrome Tungsten film.

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RESULTS Human suction blister wound healing Changes in TGFpl protein immunoreactivity are observed in human skin following injury. Normal skin contains intracellular anti-LC reactive TGFPl in the nonproliferating, differentiating keratinocytes in the suprabasal spinous and granular layer of the epidermis (Fig. lA,B). The anti-CC antibody does not stain TGFpl in normal skin (Fig. lC,D). The specificity of the antibody staining for TGFpl in skin was previously documented (Kane et al., 1990 and detailed in “Materials and Methods”) and was confirmed for all specimens in the current study as illustrated in Figure 25 and K. Immunohistochemical data on human suction blisters was obtained from a single biopsy of one individual at each timepoint. When a suction blister injury is formed in normal skin the epidermis lifts from the dermis at the basal lamina. The remaining basement membrane contains fibronectin and some laminin. The suction blister model of wound healing has been previously characterized (Mansbridge and Knapp, 1987). In contrast to the absence of anti-CC antibody staining in normal skin, anti-CC reactive TGFpl is present in the keratinocytes of the skin adjacent to the wound 24 hours after injury (Fig. 2A-C). The staining appears both intracellular and extracellular and is localized in all layers of the epidermis, including the basal cells. The basal layer

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contains the undifferentiated proliferating pool of keratinocytes in normal skin (Cowdry and Thompson, 1944; Epstein and Maibach, 1965). Cell division is transiently blocked prior to a mitotic burst in basal keratinocytes following epithelial injury (Pinkus, 1952). Directed migration is stimulated as re-epithelialization proceeds from the intact epidermal wound margin (Winter, 1962). The pattern of anti-CC reactive TGFPl changes as wound healing progresses. At 3 days post-injury TGFPl is localized primarily around the suprabasal or spinous layer keratinocytes (Fig. 2D,E). It is excluded from the basal keratinocytes which are rapidly proliferating at this stage of re-epithelialization (Pinkus, 1952). AntiCC reactive TGFpl is also significantly reduced in the keratinocytes at the original wound margin (Fig. 2F), while the staining is strong in the keratinocytes within the leading edge of the migrating epithelial sheet (Fig. 2G). At 3 days post-injury a transition is observed from principally suprabasal staining in the epidermis adjacent to the wound, to a reduction of staining at the wound margin, and to focally intense basal and suprabasal staining in the migrating epithelium within the wound. Anti-CC reactive TGFPl disappears as the wound healin process resolves, consistent with the absence of anti-C staining in normal skin. As seen at day 7 post-injury, with residual epithelial thickening and little granulosum or stratum corneum formation, antiCC TGFPl staining is reduced by comparison t o day 1 or 3 post-injury (Fig. 2H,I). Dermal staining with the anti-CC antibody has not been seen in seven normal human skin specimens (Fig. 1C,D). But anti-CC reactivity is localized in the dermis of wound healing specimens in blood vessels or in cellular infiltrates in the papillary dermis (Fig. 2). The anti-CC staining in the mesenchyme has not been associated with a specific cell type. The specificity of the antibody reactivity is demonstrated in all experiments by incubation of parallel sections with nonimmune rabbit IgG prepared in the same manner as the polyclonal antibody IgG (Fig. 2J,K). Immunohistochemistry with the anti-LC antibody reveals that the suprabasal intracellular TGFPl expression detected constitutively in normal epidermis (Fig. lA,B) is neither abolished nor relocalized following blister injury in the keratinocytes surrounding the wound throughout the wound healing process (Fig. 3). In addition to the suprabasal keratinocyte staining, basal keratinocytes at the wound margin exhibit intracellular anti-LC reactive TGF-P1 (Fig. 3C). The keratinocvtes in the mimating eDithelia1 sheet within the wouid site present iignifi”caGt1yreduced levels of antiLC TGFPl (Fig. 3D,F). Porcine partial thickness wound healing Significant differences in the immunohistochemical localization of TGFPl are observed in porcine skin and in the response of porcine skin to injury compared to human skin. It is not possible to determine whether these differences are due to variability between species or are distinct features of the experimental wounds. While applied suction causes separation at the dermal/ epidermal junction with no discernible bleeding, der-

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matome excision of a defined thickness of skin allows removal of epidermis and artial dermis with bleeding at the wound site. In adlition to re-epithelialization occurrin in the suction blister healing, healing of the partial t ickness excision injury involves dermal components directly with resynthesis of mesenchymal granulation tissue and basal lamina. Normal porcine skin exhibits a low level of intracellular anti-LC stain (Fig. 4A,B) compared to human skin, and no anti-CC stain (Fig. 4C,D) is visible in the epidermis. However, there is significant anti-CC staining in the papillary dermis of porcine skin which is not seen in normal human dermis (compare Figs. 4D and 1D). Little anti-LC staining is seen in the porcine dermis as with human dermis. There is a dramatic increase in anti-CC staining in the dermis of excised porcine skin outside the wound site as is evident 24 and 96 hours after injury (Fig. 5A-E). In the dermis underlying the wound site, the anti-CC reactive TGFpl seen in the uninjured porcine dermis is absent (Fig. 5A,C,D,F).Extracellular anti-CC reactive TGFPl is induced in the migrating keratinocytes within the wound site (Fig. 5D,F). This prominent staining is present in both basal and spinous layer keratinocytes within the wound site (Fig. 5F). The anti-CC reactive TGFpl induced in the human epidermis surrounding the blister injury-both intracellular and extracellular staining-is markedly absent in all of the porcine specimens examined from 0.5 hour to 12 days post-injury (for example, Fig. 5B,E). At 7, 10, and 12 days post-injury (Fig. 5G-L), when re-epithelialization is complete, there is no anti-CC reactive TGFPl present in the new epidermis at the wound site consistent with the resolution of the wound healing process and the absence of anti-CC staining in uninjured porcine epidermis (compare Fig. 4D). The pattern of anti-CC reactive TGFpl in the dermis outside the wound site at day 7 to day 12 (Fig. 5G-L) is comparable to the staining observed in uninjured porcine dermis while the dermal tissue underlying the wound site continues to show no anti-CC staining as seen immediately after injury (compare Fig. 5A). The pattern of anti-LC reactive TGFpl detected in normal porcine epidermis and dermis adjacent to the injury is not altered following partial thickness injury (Fig. 6A-L). The intensity of the intracellular staining is reduced in the keratinocytes migrating within the wound site (Fig. 6C,F) analogous to anti-LC TGFPl localization during re-epithelialization of the human blister (compare Fig. 3F). Induction of anti-CC reactive TGFPl in human epidermis The time-course of induction of anti-CC reactivity was evaluated in normal human skin freshly removed during myeloplasty. Anti-CC reactive TGFPl is not detected in the epidermis of an uninjured specimen as in normal skin biopsies but extracellular staining is detected in the epidermis as early as 5 minutes following incision through the epidermis and dermis. The reactivity is first seen at the incision margin and progresses away from the incision across the epidermis approximately 0.5 mm with time up to 4-6 hours post-injury (see, for example, Fig. 7A, 30 minutes

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Fig. 1. TGFpl protein immunoreactivity in normal human skin. A,B: Anti-LC antibody detects constitutive TGFpl expression uniformly in suprabasal keratinocytes in normal skin. C,D: Anti-CC antibody does not bind the form o f TGFpl present in normal skin. Features of the skin are labeled as follows: d, dermis; e, epidermis; sc, stratum corneum; g, stratum granulosum or granular layer; s, stratum spinosum or spinous layer; b, stratum basale basal layer. Magnification: A,C, X 4 2 , B,D, x 170.

post-incision). The induction of anti-CC staining is inhibited by immediate post-incision fixation (Fig. 7B) or by performing the incision in the presence of the protease inhibitor leupeptin (0.3 mgiml) (Fig. 7C) or the divalent cation chelator EDTA (1 mM) (data not shown).

DISCUSSION The dynamic changes in TGFPl expression and localization in the skin during cutaneous wound healing observed in these experimental wounds are consistent with the activity of TGFPl in in vitro systems and the series of physiological events occurring in response to injury. The scheme in Figure 8 is presented as a working model for correlation of the initiation, progression, and resolution of the cutaneous wound healing response with changes in the spatial and tem oral expression of TGFP1. Previous studies document t e in vivo localization of TGFpl protein in normal human skin and changes in its expression which occur in association with phenotypic markers of wound healing in psoriasis (Kane et al., 1990). TGFPl does not significantly alter the expression of normal keratinocyte

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differentiation in cultured cells, but it does induce the keratins characteristic of cutaneous injury in keratinocytes, sug esting that TGFPl may control expression of the woun healing phenotype (Mansbridge and Hanawalt, 1988). Keratinoc te roliferation is inhibited by TGFPl in culture (ghi$ey et al., 1986) while initiation of keratinocyte migration is stimulated (Hebda, 1988). In addition to its effects on cultured epidermal keratinocytes, in vitro TGFPl stimulates fibroblasts and endothelial cells to migrate, proliferate, and synthesize extracellular matrix (reviewed by Roberts and Sporn, 19901, essential processes to dermal repair. Changes in TGFPl immunoreactivity and localization occur in the keratinocytes and dermis adjacent to the injury and in the keratinocytes and dermis within the wound site as wound healing progresses. The very rapid and progressive induction of changes in TGFpl protein in the epidermis which propagate outward from the injury site, as seen following scalpel injury to excised human skin (Fig. 7A), suggests that TGFpl could be a primary initiator of the response to injury in the skin. TGFpl appears t o be constitutively synthe-

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Figure 2.

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Fig. 2. Changes in anti-CC TGFPl antibody reactivity during human suction blister wound healing. A-C: One day (24 hours) after injury anti-CC antibody binds a form of TGFpl induced in basal and suprabasal keratinocytes (B) adjacent to the wound and (C) at the wound margin. The wound margin is indicated with an arrowheadthe wound lies to the right. Magnification: A, X42, B,C, ~ 1 7 0D-G: . Three days after injury anti-CC staining is (E) primarily suprabasal in the keratinocytes adjacent to and continuous to at least 1.5 mm from the wound, (F) notabl absent in the keratinocytes at the original wound margin (the wounc?margin is indicated with an arrowheadthe wound lies to the right), and (G)strong in the keratinocytes in the

rapidly migrating edge of the epithelial sheet. The dark artifact in D and G is wound scab folded over the s ecimen during histochemical processing. Magnification: D, ~ 4 2E, d , x 170. H,I: Seven days after injury re-epithelialization is complete and anti-CC reactivity is reduced. Magnification: H, X42, I, ~ 1 7 0 J,K . Control nonimmune rabbit IgG prepared in parallel with the polyclonal antibodies does not react with skin sections as seen here at 3 days post-injury to human skin. Antibody specificity in skin specimens has been well documented (Kane et al., 1990; Heine et al., 1987; Flanders et al., 1989). Magnification: J, x42, K, ~ 1 7 0 .

sized in the epidermis as detected by the intracellular anti-LC antibody staining in the suprabasal keratinocytes in normal human and porcine skin. This is consistent with the isolation of TGFPl mRNA from normal skin (Elder et al., 1989). The rapid induction of anti-CC antibody reactive TGFPl may correlate with changes in the structure, conformation, or protein binding occurring during TGFPl secretion or activation in the epidermis upon injury. The blocking of this change in TGFPl by protease inhibitor (Fig. 7C) or cation chelator suggests that proteolysis or protein bindin may be involved in post-translational modification o TGFpl which rapidly alters TGFpl activity. An ordered multistep proteolytic pathway has been proposed for activation of TGFpl (reviewed by Barnard et al., 1990; Lyons and Moses, 1990) and the induction of anti-CC reactive TGFPl in skin upon injury may correspond to activa-

tion of TGFP1. There is no difference in TGFP1,2, and 3 RNA expression analyzed by in situ hybridization (unpublished observation) in parallel specimens of injured porcine or human skin in the same experimental models described here or in psoriatic human skin as previously studied (Kane et al., 1990).Thus, activation of latent protein may be more important than transcriptional activity for control of TGFpl activity in the skin. Plasmin can activate latent TGF 1 in vitro (Lyons et al., 1988) and may be responsib e for physiological activation of TGFP1. Plasminogen activators which convert plasminogen to the active protease plasmin are induced at sites of cutaneous injury (Morioka et al., 1987) providing a potential proteol tic mechanism for TGFPl activation consistent wit the rapid TGFgl changes observed upon injury to the skin. Activation of TGFpl may be the skin’s immediateresponse signal which initiates the cutaneous wound

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TGFpl IN CUTANEOUS WOUND HEALING

Fig. 3. Anti-LC antibody reactivity with constitutive TGFpl in the keratinocytes during human suction blister wound healing. A-C: One day (24 hours) after injury (B) adjacent to the wound and (C) a t the wound margin. The wound margin is indicated with a n arrowheadthe wound lies to the right. Magnification: A, ~ 4 2 B,C, , ~ 1 7 0&F: .

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Three days after injury (El adjacent to the wound. (F) Keratinocytes migrating across the blister floor during re-epithelialization do not express anti-LC detectable constitutive TGFP1. Magnification: D, X42, E,F, ~ 1 7 0G,H: . Seven days after injury re-epithelialization is complete. G: Adjacent to the wound. Magnification: G, x42, H, x170.

Fig. 4. TGFPl protein immunoreactivity in normal porcine skin. A,B: Anti-LC antibody detects a minimal level of constitutive TGFPl expression in the epidermis. C,D: Anti-CC antibody does not stain epidermal keratinocytes but does react with TGFPl in the papillary dermis. Magnification: A,C, ~ 4 2 , B,D, x170.

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Figure 5.

TGFDl IN CUTANEOUS WOUND HEALING

Fig, 5. Changes in anti-CC TGFpl antibody reactivity during porcine partial thickness wound healing. A-C: One day (24 hours) after injury anti-CC staining increases in the dermis (B) adjacent to the wound but not within the wound site. C: Migrating epithelial sheet. The wound margin is indicated with an arrowhead-the wound lies to the right. Magnification: A, ~ 4 2 , 3x, 170, C, ~ 8 5D-F . Four days (96 hours) after injury (E) dermis adjacent to the wound is anti-CC

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reactive and (F) keratinocytes migrating within the wound site are positive for anti-CC reactive TGFP1, but staining is significantly reduced in the dermis within the wound site. Magnification: D, x42, E,F, x170. Seven (G,H), 10 (I,J),and 12 days (K,L) after injury no anti-CC reactivity remains in the epidermis a t the site of injury as epidermal wound repair approaches completion. Magnification: G,I,K, ~ 4 2H,J,L, , ~170.

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Figure 6.

TGFpl IN CUTANEOUS WOUND HEALING

Fig, 6. Anti-LC antibody reactivity with constitutive TGFPl protein is constant in the keratinocytes adjacent to the injury during porcine partial thickness wound healing. Keratinocytes migrating within the wound site do not express anti-LC detectable constitutive TGFP1. A-C: One day (24 hours) after injury (B) adjacent to the wound and (Cl the migrating epithelial sheet. The wound margin is indicated with a n

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arrowhead-the wound lies to the right. Magnification: A, ~ 4 2 B, , X 170, C, x85. D-F: Four days (96 hours1 after injury (El adjacent to the wound and (F)within the wound site. Magnification: D, x42, E,F, x170. Seven, (G,H), 10 (I,J), and 12 days (K,L) after injury reepithelialization is complete. Magnification: G,I,K, x 42, H,J,L, x 170.

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polyacrylamide gel to detect antibody binding to a western blot, even though TGFpl biological activity is readily measured in the tissue extracts (personal communication, K. Flanders). Im ortantly, anti-LC and anti-CC antibodies are speci ic for purified TGFPl protein on western blots (Ellingsworth et al., 19861, in radioimmunoassay, and in enzyme-linked immunosorbent assay (Flanders et al., 1989). The specificity of anti-CC and anti-LC for distinct epitopes and conformation of the TGFPl protein is discussed in “Materials and Methods.” The specificity of anti-CC and anti-LC for TGFpl in immunohistochemistry of skin has been documented (Kane et al., 1990) and was repeated in these studies (data not shown). In summary, it is not known whether anti-CC antibody is specific for activated but not latent TGFPl in vivo as can be postulated from the immunohistochemistry and the cellular physiology. The earliest changes in TGFPl are located in both basal and suprabasal keratinocytes surrounding the wound (Figs. 2A-C, 7A). This induction of anti-CC reactive protein temporally corresponds to transient inhibition of basal keratinocyte proliferation, stimulation of keratinocyte chemotaxis, and alteration of keratinocyte differentiation and is consistent with the in vitro anti-proliferative, chemotactic, and differentiation phenotype modulation of keratinocytes by TGFPl. At later stages of healing (Fig. 2D,E) when a burst of basal keratinocyte mitotic activity is initiated, anti-CC reactive TGFpl is not present in the basal keratinocytes, perhaps representing a physiologic exclusion of active TGFPl from the proliferative basal cell population. Anti-CC reactive TGFPl is prominently expressed in the keratinocytes of the leading edge of the epithelial sheet migrating across the wound site (Fig. 2G). This localization correlates with rapid migration and synthesis of fibronectin by these cells and is consistent with the activity of TGFPl in keratinocytes in vitro, including induction of fibronectin synthesis and secretion (Wikner et al., 19881,migration of keratinocytes on fibronectin (Nickoloff et al., 19881, and promotion of chemotaxis (Hebda, 1988).There is no change in the constitutive anti-LC reactive TGFPl in the epidermis adjacent to the wound (Fig. 3B,E), but there is a dramatic reduction in anti-LC staining in the migrating keratinocytes within the wound (Fig. 3F). The anti-LC reactivity of the rotein may be altered by secretion or activation, mas ed during keratinocyte migration, or removed from the wound site by clearance pathways perhaps involving alpha-2-macroglobulin (Wakefield et al., 1988) or proteases. In addition to the suprabasal anti-LC reactive TGFPl constitutively resent, an extracellular anti-CC reactive TGFPl is etected around the suprabasal keratinocytes surrounding the injury during re-epithelialization. This extracellular TGFpl may be bound to extracellular matrix proteins or cell-surface proteins. TGFpl contains conserved matrix and integrin binding sites (Derynck et al., 1985; Pierschbacher and Ruoslahti, 1984) and TGFPl can bind fibronectin at neutral pH (Fava and McClure, 1987).TGFPl induces fibronectin synthesis and secretion by cultured keratinocytes (Wikner et al., 1988) suggesting a mechanism for TGFpl induction of extracellular matrix in the epider-

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Fig. 7. Induction of anti-CC TGFPl antibody reactivity in human epidermis. A. Anti-CC antibody staining 30 minutes after incision of excised human skin. B Inhibition of anti-CC antibody staining by immediate post-incision fixation. C: Inhibition of anti-CC antibody reactivity induction by the protease inhibitor leupeptin (0.3 mgiml). Magnification: X 170.

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a

healing athway. Characterization of the molecular weight o TGFPl which reacts with the anti-LC versus the anti-CC antibody on a western blot could in principle allow distinction of the antibody specificity for the high molecular weight latent protein versus the 25 kD active protein. However, it is not possible to load sufficient quantity of a complex tissue extract on

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NORMAL SKIN

NORMAL PRMIFERATION AND DIFFERENTIATION

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INITIATION RAPID ACTIVATION OF TGFB AT SITE OF INJURY

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- - ITHELULIUTION -MESENCHYME REGENERATW

TGFO FROM TISSUE

-INFLAMMATORY RESPONSE

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TGFPl IN CUTANEOUS WOUND HEALING

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IN SUPRABASAL AND MIGRATING KERATINWYTES. BLOOD VESSELS, AND PAPILLARY DERMIS

CHEMOTAXIS OF KERATINOCYTES, ENMlTHELlAL CELLS, FIBROBLASTS. AND IMMUNE CELLS

ACTIVITY

I

PROLlFERATlON

INITIAL PRESENCE IN BASAL KERATINOCYTES

TRANSIENT BLOCK OF KERATINOCYTE PROLIFERATION

SUBSEQUENT EXCLUSION FROM BASAL KERATINOCYTES BUT PRESENCE IN BLOOD VESSELS AND PAPILLARY DERMIS

PROLIFERATION OF KERATINOCYTES. ENDOTHELIAL CELLS, AND FIBROBLASTS

IN SUPRABASAL KERATINOCYTES ADJACENT TO INJURY

SWITCH OF KERATINOCYTE DIFFERENTIATION PATHWAY

Fig. 8. Correlation of the cutaneous wound healing response with changes in the spatial and temporal expression of TGF-P1. The physiological injury response may be described as three phases of wound healing: initiation, progression, and resolution. This working model illustrates the mechanisms by which TGFPl may coordinate the cutaneous response to injury. Physical insult may activate constitutive latent TGFpl protein at the site of injury by proteolysis or cell lysis and thus initiate the wound healing response. Progression of wound repair involves coordinate regulation of chemotaxis, prolifer-

ation, and differentiation in multiple cell types during re-epithelialization, mesenchyme regeneration, angiogenesis, and the inflammatory response. The detailed results of the immunohistochemical localization of TGFpl described in this study are presented in the context of wound healing progression and compartmentation of changes in TGFpl activity. Resolution of the wound healing response may depend on clearance of active TGFPl from the tissue by inactivation and cessation of further latent protein activation.

mis. The matrix or integrin binding of TGFpl may alter the activity of TGFPl in the skin. Localization of TGFpl in matrix may reflect physiologic compartmentalization of TGFPl activity. Alternatively, association of TGFpl with matrix or other proteins in tissue may alter the accessibility of TGFPl for activation by extracellular factors such as proteases. TGFpl also induces cells to synthesize extracellular matrix and integrins (Raghow et al., 1987; Roberts et al., 1988;Heino et al., 1989; Ignotz et al., 1989). Keratinocytes (Wikner et al., 19881, fibroblasts (Ignotz and Massague, 19861, and endothelial cells (Madri et al., 1988) synthesize extracellular matrix proteins in response to TGFP1. By controlling both extracellular matrix and cell adhesion molecules, TGFPl may control cell-to-cell and cellto-matrix attachment, thus influencing the cellular disposition to other proliferation, migration, and differentiation signals. Changes in TGFPl expression occurring in the dermis upon cutaneous injury are less complex than those observed in the epidermis. The induction of anti-CC reactive TGFPl in the human dermis following suction blister injury is localized to occasional endothelial cell clusters and regions of the papillary dermis indicating that TGFPl from platelets may be released at the site of injury and perhaps activated or deposited in the

dermis. Blood vessels in uninjured skin do not contain anti-LC or anti-CC reactive TGFPl indicating that TGFPl present constitutively in platelets (Assoian et al., 1983; Assoian and Sporn, 1986) is inaccessible to antibodies and is either not secreted or is not activated. Platelet-derived TGFpl appears to differ from TGFpl synthesized by cultured cells in that it is associated with a 125 kD binding protein (Miyazono et al., 1988) which may regulate its activity in vivo. Regions of infiltrating inflammatory cells also present anti-CC reactive TGFPl suggesting macrophage, lymphocyte, neutrophil, or eosinophil secretion or activation of TGFPl in the injured dermis. Lymphocytes and macrophages synthesize and secrete TGFpl in vitro (Roberts et al., 1986; Assoian et al., 1987).Thus, in addition to keratinocyte synthesis of TGFpl in normal epidermis, platelets or other cells resident in the human dermis may synthesize TGFpl in response to injury. In contrast to human dermis, normal porcine dermis expresses anti-CC reactive TGFpl in the papillary dermis. The staining is intensified in the dermis adjacent to the wound and absent in the dermis within the wound site. The difference may result from partial removal of dermis by excision injury in contrast to the intact dermis of the blister injury. Blood is not present in the human suction blister wound in contrast to the

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porcine partial thickness excision wound. Release of platelet-derived active TGFPl and proteases may contribute to both the increased anti-CC reactive TGFpl staining intensity in the dermis adjacent to the wound and the absence of antibody reactive protein in the dermis within the wound in the porcine partial thickness model. Subtle differences in the role of TGFPl in injury responses and wound healing may be dependent upon the presence or absence of blood-derived components within the wound. The features of TGFPl regulation of injury response and healing may differ depending on the presence of distinct clearance mechanisms in each injury or, less likely, in each species. Inflammatory response differences between the two species may account for the distinct injury responses, even though the species appear to be similar in this regard (Bouclier et al., 1990). These differences serve to emphasize the critical importance of model selection and thorough comparison prior to direct extrapolation to human physiology. In summary, TGFpl protein is constitutively synthesized in suprabasal epidermal keratinocytes in normal human skin. The structure or conformation of TGFpl is rapidly altered upon cutaneous injury as determined by anti-TGFpl antibody binding. Changes in the expression of anti-CC reactive TGFpl protein during healing are spatially and temporally regulated in a manner consistent with the activity of TGFPl on cultured cells. This is particularly important as regulation of TGFp 1 activity appears to depend primarily on activation of latent precursor protein. Anti-CC reactive TGFPl is present in migrating keratinocytes during the rapid re-epithelialization phase. TGFPl is also localized in basal keratinocytes during the time keratinocyte proliferation is transiently blocked. TGFpl is transiently associated with nonproliferating suprabasal keratinocytes during expression of the regenerative phenotype of wound repair and chemotaxis. Keratinocytes and platelets, and perhaps macrophages or 1 mphocytes, synthesize TGFPl in vivo and are stimu ated to structurally modify the TGFPl protein within a few minutes of cutaneous injury. This modification may involve association of TGFPl with extracellular proteins and perhaps activation of TGFPl from a latent form. Changes in the structure or conformation of TGFpl and changes in its localization in the skin are a very early response to injury and may initiate or coordinate the multicellular events of the wound healing response.

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ACKNOWLEDGMENTS This research was supported in part by a National Research Service Award (AR07422)from the National Institutes of Health and a Bank of America-Giannini Foundation Fellowship to C.J.M.K., by a grant (GM36617) from the National Institutes of Health to P.A.H., and by an Outstanding Investigator Award (CA44349) from the National Cancer Institute to P.C.H.

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Pietenpol, J.A., Stein, R.W., Moran, E., Yaciuk, P., Schlegel, R., Lyons, R.M., Pittelkow, M.R., Munger, K., Howley, P.M., and Moses, H.L. (1990) TGF-p1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell, 61 :777-785. Pinkus, H. (1952)Examination of the epidermis by the strip method. 11. Biometric data on regeneration of the human epidermis. J . Invest. Dermatol., 19:431447. Raghow, R., Postlethwaithe, A.E., Keski-Oja, J., Moses, H.L., and Kang, A.H. (1987) Transforming growth factor-p increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts. J. Clin. Invest., 79:1285-1288. Roberts, A.B., and Sporn, M.B. (1990) The transforming growth factor-betas. In: Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology. M.B. Sporn and A.B. Roberts, eds. Springer-Verlag, Heidelberg, vol. 9511, pp. 419-472. Roberts, A.B., Sporn, M.B., Assoian, R.K., Smith, J.M., Roche, N.S., Wakefield, L.M., Heine, U.I., Liotta, L.A., Falanga, V., Kehrl, J.H., and Fauci, A.S. (1986) Transforming growth factor type p: rapid induction of fibrosis and angiogenesis in uiuo and stimulation of collagen formation in uitro. Proc. Natl. Acad. Sci. U.S.A., 83:41674171. Roberts, C.J., Birkenmeier, T.M., McQuillan, J.J., Akiyama, S.K., Yamada, S.S., Chen, W.T., Yamada, K.M., and McDonald, J.A. (1988) Transforming growth factor p stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts. J. Biol. Chem., 263:4586-4592. Shipley, G.D., Pittelkow, M.R., Wille, J.J., Scott,R.E.,and Moses, H.L. (1986) Reversible inhibition of normal human prokeratinocyte proliferation by type beta transforming growth factor-growth inhibitor in serum-free medium. Cancer Res., 46:2068-2071. Sporn, M.B., Roberts, A.B., Shull, J.H., Smith, J.M., Ward, J.M., and Sodek, J . (1983) Polypeptide transforming growth factors isolated from bovine sources and used for wound healing in uiuo. Science, 219:1329-1331. ten Dijke, P., Hanson, P., Iwata, K.K., Pieler, C., and Foulkes, J.G. (1988) Identification of a new member of the transforming growth factor-p gene family. Proc. Natl. Acad. Sci. U.S.A., 854715-4719. Wahl, S.M., Wong, H., and McCartney-Francis, N. (1989) Role of growth factors in inflammation and repair. J. Cell. Biochem., 40:193-199. Wakefield, L.M., Smith, D.M., Flanders, K.C., and Sporn, M.B. (1988) Latent transforming growth factor-p from human platelets. J. Biol. Chem., 263:7646-7654. Wikner, N.E., Persichitte, K.A., Baskin, J.B., Nielsen, L.D., and Clark, R.A. (1988) Transforming growth factor-p stimulates the expression of fibronectin by human keratinocytes. J. Invest. Dermatol., 91:207-212. Winter, G.D. (1962) Formation of the scab and the rate of epithelialization of superficial wounds in the skin of the young domestic pig. Nature, 193t293-294.