Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia.

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.

Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair.

Transforming growth factor alpha protection against drug-induced injury to the rat gastric mucosa in vivo.

This study was designed to determine whether transforming growth factor alpha (TGF alpha) protects rat gastric mucosa against ethanol- and aspirin-induced injury. Systemic administration of TGF alpha dose-dependently decreased 100% ethanol-induced gastric mucosal injury; a dose of 50 micrograms/kg delivered intraperitoneally 15 min before ethanol decreased macroscopic mucosal injury by > 90%. At the microscopic level, TGF alpha prevented deep gastric necrotic lesions and reduced disruption of surface epithelium. Pretreatment with orogastric TGF alpha (200 micrograms/kg) only partially (40%) decreased macroscopic ethanol damage. Intraperitoneal administration of TGF alpha at a dose of 10 micrograms/kg, which does not significantly inhibit gastric acid secretion, decreased aspirin-induced macroscopic damage by > 80%. TGF alpha protection does not seem to be mediated by prostaglandin, glutathione, or ornithine decarboxylase-related events, as evidenced by lack of influence of the inhibition of their production. Pretreatment with the sulfhydryl blocking agent N-ethylmaleimide partially abolished (40%) the protective effect of TGF alpha. In addition, systemic administration of TGF alpha resulted in a two-fold increase in tyrosine phosphorylation of phospholipase C-gamma 1 and in a time- and dose-dependent increase in levels of immunoreactive insoluble gastric mucin; these events occurred in a time frame consistent with their participation in the protective effect of TGF alpha.

Particle-mediated gene transfer with transforming growth factor-beta1 cDNAs enhances wound repair in rat skin.

Based on preliminary but variable results with direct DNA transfer into wounds, we evaluated in vivo gene transfer by particle-mediated DNA delivery to rat skin to determine whether overexpression of TGF-beta1 at the site of skin incisions would result in a significant improvement in repair. Optimization of the method with viral promoter-luciferase reporter constructs indicated that expression of luciferase activity persisted up to 5 d and was promoter, pressure, and site dependent (ventral > dorsal). Using cytomegalovirus (CMV)-driven human alpha1-antitrypsin, transgene expression was immunolocalized within keratinocytes of the stratum granulosum at 24 h. We measured tensile strength of skin incisions at 11-21 d in both normal and diabetic rats transfected with TGF-beta1 expression vectors at surgery. Native murine TGF-beta1 under an SV40 promoter produced positive effects, while wound strengthening was more pronounced in diabetic animals using a CMV-driven construct. Transfection of rat skin with constitutively active, mutant porcine TGF-beta1 under the control of the CMV and Moloney murine leukemia virus promoters significantly increased tensile strength up to 80% for 14-21 d after surgery. Transfection 24 h before surgery was more effective. Particle-mediated gene delivery can be used to deliver viral promoter-cytokine expression constructs into rat skin in a safe, efficient, and reproducible fashion. The extent of wound repair, as evidenced by enhanced tensile strength, can be markedly improved in tissues transfected with TGF-beta1 expression constructs.

Mechanical abrasion improves early incorporation of small intestinal submucosa.

It has been shown that gross incorporation of porcine-derived small intestinal submucosa (SiS) is limited at 2 weeks. This study evaluates a technique for improving the early incorporation of implanted eight-ply SiS. Six pigs underwent implantation of SiS on the peritoneal surface using three techniques: suture fixation of stock-perforated SiS, suture fixation of manually perforated SiS, and suture fixation of stock-perforated SiS to mechanically abraded peritoneum. Gross incorporation was evaluated and random samples harvested for tensiometric analysis 2 weeks after implantation. SiS placed onto mechanically abraded peritoneum demonstrated significantly greater gross incorporation than both stock-perforated SiS (100% versus 42%, P = 0.015) and manually perforated SiS (100% versus 50%, P = 0.042). There was no difference in gross incorporation between stock and manually perforated SiS. Using tensiometric analysis, the force required to separate the peritoneum from the SiS implant was significantly greater for the SiS placed onto mechanically abraded peritoneum (4.4 +/- 1.7 kg . f/cm2) than for both the stock-perforated SiS samples (1.0 +/- 0.5 kg x f/cm2) and the needle-perforated SiS samples (1.4 +/- 0.9 kg x f/cm2; P < 0.001). There was no difference between stock and manually perforated SiS at 2 weeks. Mechanical abrasion of the peritoneum before SiS onlay leads to improved gross incorporation 2 weeks after implantation in a porcine model of herniorrhaphy. Long-term studies and histologic analysis are needed to validate this method as a means for improving early incorporation of SiS.

The central ACL defect as a model for failure of intra-articular healing.

Intra-articular soft tissues, such as the anterior cruciate ligament (ACL), fail to heal in contrast to the extra-articular medial collateral ligament (MCL), which undergoes classic healing. The goal of this study was to validate a model for failure of intra-articular healing that could be used in the future to test new repair strategies. We conducted a two-part experiment, the first part ex vivo, and the second in vivo. Our initial ex vivo experiments were used to determine the optimal width of the central defect in the canine ACL that would produce reproducible structural properties at time zero. The second experimental series used this optimal scalpel blade width to create a central defect in the canine ACL followed by measurement of structural properties in the ACL after either a 3- or 6-week in vivo healing period. A 3.5-mm beaver blade resulted in a maximum tolerated load of 56.8 +/- 4.7% (mean +/- SEM) of control at time zero. After the 3- and 6-week in vivo healing periods, the maximum load was 74.6 +/- 5.3 at 3 weeks and 64.9 +/- 3.8% at 6 weeks compared to control. Thus, biomechanical parameters tested at 6 weeks after creation of a defect showed no significant gains from defects tested immediately after the creation of injury. The centrally placed ACL defect in this canine model demonstrates failure to mechanically heal, which should prove suitable for future in vivo evaluation of the biomechanical and histological response to tissue engineering repair strategies for intra-articular soft tissues.

Concurrent overexpression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4) in intestinal adenomas from multiple intestinal neoplasia (Min) mice and human familial adenomatous polyposis patients.

We postulated that increased expression of the cell cycle regulators cyclin D1 and cyclin-dependent kinase (Cdk) 4 may be involved in the development of intestinal adenomas associated with familial adenomatous polyposis (FAP). In the present study of multiple intestinal neoplasia (Min) mice and human FAP patients, the expression and distribution of cyclin D1, Cdk4, and cell proliferative activity (5-bromo-2'-deoxyuridine incorporation) in normal and adenomatous intestinal epithelium were investigated. Immunohistochemical analysis of Min mouse intestine revealed that cyclin D1 immunoreactivity in the intestinal epithelium was restricted to the adenomatous areas, with a significantly higher percentage of positively staining nuclei in high-grade dysplasia versus low-grade dysplasia (54.8 +/- 18.4% versus 34.6 +/- 16.9%, P = 0.016). Morphologically normal areas of intestinal epithelia were uniformly negative for cyclin D1 immunoreactivity. Cdk4 nuclear immunoreactivity was restricted to the crypt areas in morphologically normal small intestine and colon. Conversely, Cdk4 immunoreactivity was uniformly abundant in adenomatous areas regardless of the degree of dysplasia. Increased expression of cyclin D1 and Cdk4 in adenomas was accompanied by a significantly increased 5-bromo-2'-deoxyuridine incorporation rate in the same areas. Immunoblot analysis of lysates from surgical specimens revealed increased levels of cyclin D1 and Cdk4 in the majority of intestinal adenomas from human FAP patients in comparison to the adjacent grossly normal colonic mucosa. Our results indicate that overexpression of cyclin D1 and Cdk4 occurs in intestinal adenomas and is associated with increased cell proliferative activity in premalignant neoplastic cells. Increased cyclin D1 immunoreactivity is associated with more severe dysplasia. These data suggest that abnormal up-regulation of these important G1 cell cycle proteins is a relatively early event in intestinal carcinogenesis and that these changes may contribute to malignant progression within those lesions.

Decreased transforming growth factor beta type II receptor expression in intestinal adenomas from Min/+ mice is associated with increased cyclin D1 and cyclin-dependent kinase 4 expression.

Tumor cells often become resistant to the growth-inhibitory effects of transforming growth factor beta (TGF-beta). Recent studies have identified TGF-beta type II receptor (RII) mutations in a subset of cancers, including colon cancer. To evaluate the expression of TGF-beta RII in premalignant intestinal adenomas and the relationship with cell cycle regulation, we investigated the expression of TGF-beta RII, cyclin D1, and cyclin-dependent kinase 4 (Cdk4) in Min/+ mouse intestinal adenomas. Immunohistochemistry indicated that TGF-beta RII cytoplasmic immunoreactivity was undetectable in the proliferative crypt zones of the normal small intestinal and normal colonic epithelium but was abundant toward the villus tips of the normal small intestine and the lumenal third of the colonic glands. As was observed in the proliferating crypt zones, TGF-beta RII immunoreactivity was dramatically decreased or undetectable in all adenomas examined in comparison to the abundant levels in adjacent normal differentiated intestinal epithelium. TGF-beta RII mRNA was also reduced in the adenomas in comparison to normal mucosa as determined by reverse transcription-PCR. In an inverse distribution to TGF-beta RII, Cdk4 nuclear immunoreactivity was restricted to the crypt regions of the small and large intestine, whereas cyclin D1 immunoreactivity was uniformly absent in normal intestinal epithelium. For both cyclin D1 and Cdk4, protein and mRNA levels were increased in intestinal adenomas but not in normal intestinal epithelium as determined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. In summary, the lack of TGF-beta RII expression was associated with increased cyclin D1 and Cdk4 expression in Min/+ mouse intestinal adenomas. We hypothesize that the former may enable tumor cells to escape from the normal growth-constraining influence of TGF-beta, whereas the latter promotes inappropriate cell proliferation and adenoma progression.

Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice.

Cyclin D1 is a known oncogene and a key regulator of cell cycle progression. Amplification of the cyclin D1 gene and its overexpression have been associated with aggressive forms of human hepatocellular carcinoma (HCC). In this study, two independent lines of transgenic mice have been generated that express cyclin D1 under the control of the rat liver fatty acid binding protein promoter. This transgene specifically directs expression in the liver and the intestines. RNA and protein analysis demonstrated increased expression of the cyclin D1 gene product in the liver and bowel when compared with wild-type siblings. Both transgenic lines developed progressive liver disease. Examination of H&E stained sections of the liver and bowel revealed hyperplastic changes in the liver by 3 months of age. By 6 months of age, transgenic mice had obvious hepatomegaly and histological evidence of dysplasia in the liver. These early changes were significantly more dramatic in male animals when compared with female animals. By 9 months of age adenomas of the liver appeared, progressing to HCC over the ensuing 6-month period. By 15-17 months of age, 87% of male and 69% of female animals had either adenomatous nodules or HCCs. By 17 months of age, 31% of male and female animals had disease that had progressed to HCC. These animals represent a unique and significant new model for the study of human HCC. This study demonstrates that overexpression of cyclin D1 is sufficient to initiate hepatocellular carcinogenesis.

The tumorigenic and angiogenic effects of MGSA/GRO proteins in melanoma.

Continuous expression of the MGSA/GROalpha, beta, or gamma chemokine bestows tumor-forming capacity to the immortalized murine melanocyte cell line, melan-a. The mechanism for this transformation is unclear, although both autocrine and paracrine processes are possible because melan-a cells as well as endothelial cells express a low level of the receptor for this ligand. To further define the role of MGSA/GRO proteins in melanocyte transformation, two types of experiments were designed to neutralize the biological effects of MGSA/GRO in the transfected melan-a clones: (1) the effect of neutralizing antiserum to MGSA/GRO proteins on melan-a tumor growth was assessed; (2) the tumor-forming capacity of melan-a clones expressing ELR motif-mutated forms of MGSA/GRO with compromised receptor affinity was compared to the tumor-forming capacity of clones expressing wild-type MGSA/GRO. These experiments revealed that SCID mice inoculated with MGSA/GROalpha- or gamma-expressing melan-a cells and subsequently treated with antiserum to the respective chemokine exhibited decreased tumor growth. This reduction in tumor growth was accompanied by declining angiogenic activity in MGSA/GROgamma-expressing tumors. Moreover, athymic nude mice injected with melan-a cells expressing ELR-mutant forms of MGSA/GROalpha exhibited markedly impaired tumor-forming capacity compared with those mice injected with melan-a clones expressing wild-type MGSA/GRO. These data suggest that continuous expression of MGSA/GRO proteins may facilitate tumor growth by stimulating the growth of microvessels into the tumor (paracrine) and by affecting melanocyte growth (autocrine).

The effects of severe thermal injury on the glycosaminoglycans of guinea pig skin.

The central purpose of the present investigation was the qualitatively and quantitatively analyze the glycosaminoglycans of guinea pig skin at 2 hr after a third-degree burn injury. The data obtained from uronic acid determinations, fractionation by column chromatography and cellulose acetate electrophoresis indicate no significant alterations in the glycosaminoglycans at 2 hr postburn. The data presented in this study support the concept that the glycosaminoglycans are not immediately damaged by the heat of the burn.

Epidermal and dermal effects of epidermal growth factor during wound repair.

Epidermal growth factor (EGF), a well-characterized peptide that stimulates in vitro cell proliferation, has now been shown to enhance in vivo resurfacing of porcine wounds. Topical formulations containing either recombinant EGF or placebo were applied daily to partial-thickness wounds along the dorsal surface of pigs. Following full-thickness removal of these wounds, tissues were sectioned and stained, and histologic sections were subjected to computerized morphometric analysis. A significant acceleration of epithelialization across the wound surface was noted following daily EGF treatments. EGF delivered in a variety of topical formulations also produced a marked increase in the cellularity and thickness in the neodermis. A dose-responsive increase in the thickness of the granulation tissue was also observed. In conclusion, topical application of EGF stimulates epithelialization of partial-thickness wounds and produces a positive impact on the underlying dermis during the early phases of wound repair.

Immunolocalization of collagenase and TIMP in healing human burn wounds.

Degradative events in remodeling connective tissues are mediated through the actions of one or more members of the matrix metalloproteinase family. Conversely, members of the tissue inhibitors of metalloproteinase (TIMP) family act to attenuate proteolysis. Because collagenase and TIMP are rapidly secreted into the extracellular matrix following their biosynthesis and may not remain near their cell of origin, we undertook an immunohistochemical examination of human burn injuries to establish the distribution of these proteins during acute wound repair. Immunostaining for collagenase and TIMP was markedly increased within the wound bed but not in adjacent regions of histologically normal skin. Immunoreactive collagenase was first noted at the eschar-dermal interface by day 3 after injury and became very prominent in the dermis from day 5 to day 17. By day 5, focal patches of immunoreactive collagenase were found at the epidermal-dermal junctions at the wound margins. Within the wound bed, intense staining for collagenase was noted in the connective tissue surrounding the surviving epithelial appendages and around blood vessels. Immunoreactive TIMP was detected by day 2 both in the dermis and the overlying eschar but rapidly assumed the same interfacial pattern as described for collagenase. Staining for TIMP was only sporadically found at the dermal-epidermal margins and surrounding surviving epithelial appendages. Like collagenase, TIMP was prominently localized about vascular structures. These studies demonstrate that, in acute wounds, immunoreactive collagenase and TIMP are generally increased throughout the area of injury but particularly so at interface zones including eschar-dermis, epidermis-dermis, appendages-dermis, and around vascular structures.

Localization of mRNAs representing interstitial collagenase, 72-kda gelatinase, and TIMP in healing porcine burn wounds.

The process of wound healing sets in motion a complex and dynamic series of events, which includes the remodeling of the extracellular matrix. Degradation of matrix macromolecules is mediated through the actions of the matrix metalloproteinase family. Conversely, the actions of this enzyme family are regulated by tissue inhibitors of metalloproteinases (TIMPs). In this study, we have developed riboprobes derived from human cDNAs representing collagenase, 72-kDa gelatinase, and TIMP and have found them to be sufficiently specific and sensitive for use in in situ hybridization studies of porcine burn wounds. Expression of these mRNAs, although not seen in uninjured skin, was found to be a predictable and locally distinct event in wound repair. Transcripts for collagenase and TIMP but not 72-kDa gelatinase were detected at the resurfacing epithelial margin; label was also detected in and around follicular epithelium within the wound bed. Transcripts for both metalloenzymes and TIMP were found throughout the viable dermis and subcutaneous tissues underlying the wound bed. However, expression of 72-kDa gelatinase was most prominent in the superficial dermis adjacent to the resurfacing epidermis at the wound margin. Collagenase and TIMP transcripts were particularly prominent in a perivascular pattern in the dermis and in the connective tissue network surrounding adipocytes in the subcutaneous zone. Numerous cell types appeared to be involved, including keratinocytes, fibroblasts, macrophages, and endothelial cells. Future exploitation of this porcine thermal injury model is likely to provide information about the spatial and temporal patterns of matrix metalloproteinase and TIMP expression in cutaneous wound healing.

Annexin-1 localization in human skin: possible association with cytoskeletal elements in keratinocytes of the stratum spinosum.

Annexin-1 (also called lipocortin-1 or p35), a putative substrate of the epidermal growth factor/receptor kinase, protein kinase C, and transglutaminase, was immunolocalized in embryonic, neonatal, adult, and diseased human epidermis. In embryonic skin intense annexin-1 immunoreactivity was found in the periderm at 54 d estimated gestational age (EGA). Later (EGA = 91-143 d), annexin-1 immunoreactivity was restricted to basal keratinocytes. In neonatal skin, basal cells were often more heavily stained than were suprabasal keratinocytes, which were also stained. Only basal keratinocytes stained in adult plantar skin, but in thin skin annexin-1 was present in the basal, suprabasal, and sometimes even in the granular layers of the epidermis. Often, annexin-1 appeared concentrated around the perimeter of cells, especially tonofilament/desmosome-rich keratinocytes of the spinous-cell layer. At high magnification, annexin-1 appeared associated with distinct structures and was very granular in appearance in the intensely stained ductal keratinocytes of eccrine sweat glands, cells that are very highly enriched in keratin tonofilaments. This striking distribution in certain keratinocytes enriched in tonofilaments suggests a role for annexin-1 in cytoskeletal functions.

Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin: incisional wound model.

Enhanced wound healing is elicited by exogenous administration of transforming growth factor- beta 1 (TGF- beta 1) in split-thickness, excisional wounds in the pig (Quaglino, Lab Invest 63:307-319, 1990). A study was designed to investigate if the selective and localized effects of TGF-beta 1 found in the previous model were dependent upon the type of wound or could be considered a more general effect of the cytokine. Transdermal, sutured incisions in the pig were evaluated by conventional histology and by in situ hybridization to reveal locally affected gene expression of collagen, elastin, fibronectin, stromelysin, TGF- beta 1, and basic fibroblast growth factor. Granulation tissue formation was markedly enhanced at 6 d by a single injection of recombinant human TGF beta 1 at the time of wound closure. Although granulation tissue was confined within the margins of the incisional wound, prominent differences in hybridization signals were observed between control and treated wounds. The stimulatory effect of TGF- beta 1 on granulation tissue formation was accompanied by a distinct enhancement in cells expressing mRNA for several different extracellular matrix proteins including collagens type I and III and elastin, whereas a single injection of human recombinant TGF beta 1 (4 micrograms) at the wound site diminished the expression of the neutral metalloprotease, stromelysin, and enhanced the frequency and intensity of cells expressing TGF- beta 1. The data reinforce the concept that TGF- beta 1 can act as a potent, auto-inductive modulator of connective tissue remodeling during the repair process.

Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis.

Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

Modulation of epidermal growth factor receptors in psoriatic lesions during treatment with topical EGF.

Active psoriatic lesions have increased EGF/TGF alpha receptors, historically known as the EGF-R. This increase is due to their persistence into the outer parakeratotic layers as measured by autoradiography, immunohistochemistry, and mRNA assays. When psoriatic lesions in patients resolve due to therapy with different modalities, the EGF-R persistently expressed in the outer layers of the epidermis either disappear or resume a basal location presumably due to receptor downregulation. To test whether EGF could downregulate EGF-R and biologically affect psoriatic epidermis, split-thickness skin grafts of active psoriatic lesions were sutured onto the dorsal surface of nude mice. After 3 weeks, the mice were treated daily for a 6-week period with placebo, or 10 or 50 micrograms/ml EGF. Immunostaining showed persistent EGF-R in all epidermal layers in the untreated, placebo-, and 10 micrograms/ml EGF-treated groups. Those grafts receiving a high dose of EGF (50 micrograms/ml) showed either no immunoreactive EGF-R or faint basilar staining. As an additional check for functional activity of the EGF-R, an abundant substrate for this receptor, PLC-gamma 1 was also evaluated following EGF treatment. A similar distribution and modulation pattern following treatment were observed in the grafts immunostained for PLC-gamma 1, suggesting that exogenous EGF treatment affected metabolic pathways subsequent to ligand receptor binding. Morphologic alterations characteristic of a regressing psoriatic phenotype (a decrease in acanthosis, thickness, and the resumption of the orthokeratotic mode of differentiation) were noted in those lesions receiving the 50 micrograms/ml EGF treatment. This study indicates that persistent EGF-R in psoriasis vulgaris are biologically active in vivo and may serve a pivotal role in the regulation of psoriatic lesions.

Quantitative determination of EGF-R during epidermal wound healing.

Little is known about the intrinsic regulation of growth factors of cytokines during the normal epidermal wound-healing processes in skin. A simplified model of wounding (tape stripping to remove the stratum corneum) was used to study the role of epidermal growth factor receptors (EGF-R) in this process. Although the dynamics of EGF-R in epidermal wound healing have not been determined, the immunoreactive EGF-R that are present presumably play an active role. Prior studies show that 1) EGF-R are present in increased numbers in proliferative skin diseases; 2) a hypertrophic epidermis, closely resembling normal wound healing, is induced in mouse skin by EGF injections; and 3) exogenous topical EGF potentiates wound healing. The number of immunoreactive receptors as measured by an enzyme-linked immunosorbent assay (ELISA) and histologic methods increased prior to an increase in epidermal thickness, total protein, and DNA content. This early increase in the levels of EGF-R was followed by a sharp decline in EGF-R and subsequent decline in epidermal thickness (hypertrophy), total protein, and DNA levels. Alterations in the temporal sequence in these parameters indicate that the EGF-R-mediated signaling systems play an active role in epidermal wound repair.

Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages.

To localize epidermal growth factor (EGF) receptors in normal human epidermis and other skin structures, two different light microscopic methods were used. EGF binding [( 125I]EGF/R) to the extracellular portion of the EGF receptor was studied by incubating intact skin samples with [125I]EGF, sectioning the tissues, and performing autoradiography. Immunoreactive EGF receptor molecules (IR-EGF/R) were localized with a mono-specific anti-EGF receptor antibody using a 2-step indirect immunocytochemical method (horseradish peroxidase) and detergent permeabilized tissues. This latter method measured the total pool of EGF receptors: occupied and/or internalized forms, precursor forms, and partially degraded forms of the EGF receptor that retain immunoreactivity. Both the [125I]EGF/R and IR-EGF/R localization studies indicated that EGF receptors were present in basal epidermal keratinocytes, sebocytes, outer root sheath cells in hair follicles, smooth muscle cells of arrector pili muscles, and dermal arteries. The highest levels of [125I]EGF/R and IR-EGF/R were found in the dermal ducts of eccrine sweat glands. The distribution of both [125I]EGF/R and IR-EGF/R was not consistent with the concept that EGF exclusively is involved in cellular division and proliferation in normal human epidermis and its appendages, i.e., EGF receptors were also found in tissues that do not undergo rapid proliferation. The present study indicates that EGF may have a more complex regulatory role in the skin than was previously thought.