Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes.

Antibodies which bind to different nuclear antigens in tissue sections or in permeabilized cell cultures are useful markers of subsets of connective tissue disease, especially of lupus erythematosus (LE), but whether these antibodies are able to react with these intracellular sequestered antigens in vivo and cause immunologic tissue damage has been a matter of much debate. We report experiments which show that ultraviolet light-irradiated, cultured human keratinocytes bind IgG antibodies from the sera of LE patients with either monospecific anti-SSA/Ro, anti-RNP, or anti-Sm activity, which implies that these antigens have been made accessible on the cell surface by ultraviolet irradiation. Normal human sera or LE patient's sera with anti-double-stranded DNA, anti-single-stranded DNA, or antihistone activity do not bind to the surface of irradiated human keratinocytes. In control experiments, all antisera produced the expected patterns of nuclear and cytoplasmic staining of fixed permeabilized, unirradiated keratinocytes. Careful study of the viability and permeability of irradiated keratinocytes by several techniques showed that this apparent cell membrane expression of extractable nuclear antigens (SSA/Ro, RNP, and Sm) following irradiation was seen on injured keratinocytes whose cell membranes were intact, but not on dead cells. It is particularly significant that all six monospecific anti-SSA/Ro sera bound to irradiated keratinocytes, since this antibody antigen system is highly associated with photosensitive cutaneous LE.

Epithelial polymeric immunoglobulin receptors.

The secretory immune system, which leads to secretion of polymeric immunoglobulins along mucosal surfaces, has not been shown to have any definite role in cutaneous immunology, although the polymeric immunoglobulin receptor, secretory component (SC), has been found in sweat glands and possibly in the epidermis. The purpose of this study is to examine normal human skin and cultured human keratinocytes for the presence of SC. Positive staining for SC was found in sections of normal human skin along the basement membrane zone with use of a polyclonal antibody to SC and focally on the surfaces of epidermal cells with use of a monoclonal antibody to SC. Granular cell-surface fluorescence of an intensity far less than that of the positive control HT 29 cells was seen when cultured human keratinocytes were stained for SC by indirect immunofluorescence (IF). Study of lysates of both HT 29 cells and HK by immunoblotting have been negative, perhaps due to destruction of the protein or loss of antigenicity during the extraction process. If human keratinocytes are capable of expression of SC, and the receptor can interact with IgA and IgM, this might be a mechanism for protection of the skin from microbial agent or foreign antigens and might be relevant to the deposition of IgA seen in certain skin diseases.

Serum-free serial culture of adult human keratinocytes from suction-blister roof epidermis.

Coating cell culture flasks with natural extracellular matrix (ECM) enhanced the culture of adult human keratinocytes from suction-blister roof epidermis in an environment without fetal calf serum (FCS), bovine pituitary extracts or cellular feeder layers. A higher incidence of cell attachment on natural ECM was observed than on collagen and human fibronectins (HFN)-coated plastic dishes, and natural ECM was necessary for growth and proliferation of attached cells under the culture conditions used. Cells in primary culture grew to confluency on natural ECM-coated surfaces within about 14 days, and subsequent serial passage could be made up to fourth passage in collagen- and HFN-coated plastic flasks. Cultured keratinocytes in this serum-free environment formed colonies of small cuboidal, healthy cells with little keratinization or stratification and demonstrated antigenic characteristics of human basal cells.

Detection of herpes simplex virus DNA in cutaneous lesions of erythema multiforme.

The association between erythema multiforme (EM) and herpes simplex virus (HSV) infection has long been appreciated, although the exact role which HSV may play in the pathogenesis of this herpes-associated EM (HAEM), is unknown. Previous studies have suggested, but not definitively demonstrated, the presence of HSV in lesions of HAEM. The presence of HSV would support the hypothesis that an immune-mediated response directed against HSV-specific antigens in the skin is central to lesion development in HAEM. The purpose of this study was to examine lesions of EM for the presence of HSV DNA by using the polymerase chain reaction (PCR). In addition, in situ hybridization using an HSV-specific RNA probe was performed to further localize the HSV nucleic acids within the skin. DNA was extracted from formalin-fixed, paraffin-embedded specimens of cutaneous lesions of HAEM and also from EM for which no precipitating factor could be documented, otherwise known as idiopathic EM (IPEM). DNA from lesions of bullous pemphigoid served as a negative control. Using PCR to specifically amplify HSV sequences which might be present, and then performing Southern analysis, we demonstrated HSV DNA in 9/13 HAEM and 6/9 IPEM biopsies. No HSV was detected in six lesions of bullous pemphigoid. In situ hybridization of three cutaneous HAEM lesions using an 35S-labeled HSV-specific RNA probe localized the HSV nucleic acids predominantly to the epidermis. Three biopsies of chronic dermatitis, used as negative controls, did not demonstrate this specific hybridization. These findings confirm the presence of HSV in lesions of HAEM and are consistent with the concept of an HSV-specific immune-mediated pathogenesis for this disease. In addition, most cases of IPEM appear to be herpes associated despite the absence of clinically apparent HSV infection.

Wheat protein antibodies in dermatitis herpetiformis.

An enzyme-linked immunosorbent assay was used to detect class-specific antibodies to wheat protein antigens. Antibodies which we detected by this technique reacted indistinguishably with antigens prepared from crude gluten, crude gliadin, alpha-gliadin, Frazer fraction III, and subfraction B and B3 of Frazer fraction III. No sera reacted with a human serum albumin control antigen. The prevalence of IgG antibodies to wheat protein antigens was significantly greater in patients with gluten sensitive enteropathy, 12 of 17, (p = .00011) and in patients with dermatitis herpetiformis, 5 of 14, (p = .046) than in normal control subjects. Strongly positive reactions for IgG antibodies were present only in patients with gluten sensitive enteropathy or dermatitis herpetiformis. IgA antibodies to wheat protein antigens were found only in gluten-sensitive enteropathy patients. We have found this to be a sensitive, precise technique for measurement of antibodies to wheat protein antigens and feel that it will prove useful in evaluation of the role of immune complexes involving wheat protein antigens and their antibodies in the pathogenesis of dermatitis herpetiformis.

Enhancement of specific immunofluorescent findings with use of a para-phenylenediamine mounting buffer.

A recently described immunofluorescence mounting buffer containing para-phenylenediamine prevents fading of specific staining in skin sections during microscopic examination, and allows better appreciation of morphological detail. Examination of slides at high powers with intense illumination, as well as improved photomicrographs, are possible with this reagent.

Detection of gluten in human sera by an enzyme immunoassay: comparison of dermatitis herpetiformis and celiac disease patients with normal controls.

We have developed a triple sandwich enzyme immunoassay to detect circulating gluten in human sera. With human sera containing known amounts of added gluten as controls, the assay was sensitive in the range of 0.75 to 75 micrograms of gluten per ml of serum. Forty-one control subjects were compared to 21 patients with dermatitis herpetiformis and 11 patients with celiac disease. The dermatitis herpetiformis and celiac disease patients had significant elevation of serum gluten values over the control subjects. Circulating gluten antigenemia is a previously unrecognized feature which may be important in understanding the pathogenesis of dermatitis herpetiformis and celiac disease.

Basal cell carcinomas grown in nude mice produce and deposit fibronectin in the extracellular matrix.

Epidermal cells in vitro produce and deposit fibronectin (FN) in the pericellular matrix. Such FN production by epidermal cells may be involved in vivo in wound reepithelialization, tissue morphogenesis, and growth of epithelial tumors. The purpose of this study was to examine whether the FN, previously shown to be within and surrounding human basal cell carcinoma (BCC) lobules, was in part the product of epidermal-derived tumor cells. To examine this question we took advantage of our ability to grow human BCC in nude mice. Since we could demonstrate that all stromal cells surrounding the BCC were of mouse origin, antibodies specific for human FN would distinguish epithelial-derived FN from mesenchymal-derived FN. Five solid BCCs were implanted subcutaneously in nude mice. Growing tumors were removed after 60 days, snap-frozen, sectioned on a cryostat, and verified microscopically as BCC. The Hoescht DNA stain, which can distinguish mouse and human nuclei, demonstrated that mouse, not human, fibroblasts occupied the stroma surrounding each tumor lobule. Sections of all 5 BCCs were stained by immunofluorescence and immunoperoxidase techniques with antibodies to bullous pemphigoid (BP) antigen, laminin (LM), and FN. BP antigen and LM were present at the basement membrane zone (BMZ) of all tumor lobules as previously described for in situ BCC. FN staining was present along the BMZ, within the tumor lobules, and in the surrounding stroma. Antibodies to human FN were passed over a mouse FN affinity column to absorb antibodies which cross-reacted with mouse FN. The resultant antibody preparation, which was specific for human FN in this system, continued to demonstrate FN along the BMZ and within the tumors, but failed to stain FN in more distant stroma. Epidermal-derived cells, therefore, can synthesize and deposit FN in vivo in adjacent extracellular matrix. We speculate that this FN matrix may facilitate growth of BCC in this model.

Proliferating cells of human basal cell carcinoma are located on the periphery of tumor nodules.

Study of the growth characteristics of basal cell carcinoma (BCC), a relatively well-organized, slow-growing skin cancer, has been limited because of the lack of methods for propagation of the tumor off the human host. We have used newly developed techniques for transplantation and propagation of BCC on athymic mice to study [3H]thymidine incorporation by nodular BCC. In human BCCs labeled in vitro immediately after removal from the mice and in vivo on the mice, [3H]thymidine during a 4-h pulse was incorporated primarily by cells on the periphery of tumor nodules (labeling indices 6-24%) rather than by the cells more central in tumor nodules (labeling indices 0-2%). Similar results were also seen when samples of tumor freshly removed from patients were labeled in vitro. We conclude that the dividing cells within nodular BCC are primarily the cells at the edges of tumor nodules and that this characteristic is related to the slow, progressive, invasive growth of BCC.

Characterization and practical benefits of keratinocytes cultured in strontium-containing serum-free medium.

Strontium (Sr2+) can substitute for Ca2+ and stimulate a variety of functions of numerous types of cells. The purpose of this study was to investigate the details of the biologic effects of Sr2+ on human keratinocyte growth, cell cycle, viability, and differentiation and to compare these effects with Sr2+ effects on cultured skin melanocytes. Cultured keratinocytes stimulated with 1.0-3.0 mM Sr2+ showed higher viability and almost a twofold increase in cell number compared with those grown in a standard calcium concentration. Time course studies revealed that 2.0 mM Sr2+ had no effects on growth of cultured melanocytes or fibroblasts. Sr2+ increased the percentage of cultured keratinocytes in G2/M phase, with a decrease in cells in G0/G1 phase. This effect of Sr2+ on the cell cycle was not seen in cultured melanocytes or fibroblasts. A 2 mM concentration of Sr2+ produced an increase in low-density keratinocytes separated by a Percoll gradient. In addition, increased expression of human fibronectin was observed in the cytoplasm and on cell membranes of keratinocytes cultured in Sr2+. Sr2+ can be of practical benefit in the culture of human keratinocytes in serum-free medium, increasing the viability and proliferative rate and producing a more uniform population of basaloid cells with increased expression of cell surface fibronectin.

Class-specific antibodies to gluten in dermatitis herpetiformis.

An immune reaction to wheat protein has been previously proposed to explain the pathogenesis of dermatitis herpetiformis. In order to detect and characterize antibodies to gluten in human sera, we developed an enzyme immunoassay for class-specific antibodies. Results of this assay in 49 patients with dermatitis herpetiformis were compared with those of 38 normal control subjects, 11 patients with celiac disease, and 6 small-bowel bypass patients. IgA antibodies to gluten were significantly more frequent in dermatitis herpetiformis sera (28/49) than in normal control sera (4/38). IgG antibodies to gluten were significantly more frequent in both celiac disease (10/11) and dermatitis herpetiformis (16/49) sera than in control (5/38) sera. Dermatitis herpetiformis sera also had an increased prevalence of IgM antibodies to gluten (19/49). Small-bowel bypass patients demonstrated no antibody to gluten. Antibodies to gluten in dermatitis herpetiformis objectively mark a state of immune reactivity to wheat protein and may be involved in the genesis of the cutaneous IgA immune deposits and the skin disease.

Fibronectin in basal cell epithelioma: sources and significance.

Nodular basal cell epitheliomas (BCE) contain fibronectin both within tumor nodules and at the nodule-stroma interface (basement membrane zone). Fibronectin within or at the periphery of tumor nodules could be derived from the tumor cells, from entrapped stroma, or from plasma. The present study was designed to elucidate the source(s) of fibronectin within BCE nodules. If stromal entrapment occurred to any great extent, von Willebrand factor VIII:Ag-stained blood vessels within tumor nodules should be evident by immunofluorescence techniques. Likewise, if plasma proteins were deposited in BCE, the tumor nodules should stain with fluorescein-conjugated antifibrinogen antibodies. Therefore, 6 basal cell epitheliomas were double labeled with rhodamine-conjugated antihuman fibronectin and fluorescein-conjugated antihuman factor VIII:Ag or fluorescein-labeled antihuman fibrinogen. Fibronectin was present in a linear disposition along the margin of tumor lobules and as a fine filamentous deposit in the central portions of tumor tissue. There was no evidence of fibrinogen or factor VIII:Ag in any of the tumor lobules. Factor VIII:Ag was present in a granular pattern within blood vessel walls that coursed between tumor nests. An indirect immunoperoxidase technique using rabbit antihuman fibronectin and peroxidase-labeled goat antirabbit IgG demonstrated that fibronectin within the central portion of the tumor lobules was closely associated with the tumor cells. The absence of fibrinogen and factor VIII:Ag within the tumor tissue indicates that the fibronectin is probably not plasma- or stroma-derived while immunoperoxidase data suggest that fibronectin may be a product of BCE cells.

The demonstration of SS-A/Ro antigen in human fetal tissues and in neonatal and adult skin.

Anti-SS-A/Ro antibodies have been statistically associated with congenital heart block and cutaneous lupus in the neonatal lupus syndrome, and with photosensitive cutaneous lupus in subacute cutaneous lupus erythematosus, but it had not been demonstrated that SS-A/Ro antigen is present in fetal tissues or at any age in human skin. We examined normal fetal tissues, normal neonatal and adult skin, and keratinocytes from purified serum-free cultures by immunofluorescence (IF) and by immunodiffusion or counterimmunoelectrophoresis (CIE) to determine the presence of SS-A/Ro antigen. We found SS-A/Ro antigen to be present in fetal hearts and in other internal organs. SS-A/Ro antigen could be demonstrated in biopsies of neonatal and adult skin by IF and was confirmed to be in keratinocytes by CIE. SS-A/Ro antigen may be found on the surface of keratinocytes in culture and therefore may be present at a relevant site for antibody binding. We have shown that SS-A/Ro antigen is a normal component of tissues that may be affected in neonatal lupus (fetal myocardium, neonatal epidermis) and in subacute cutaneous lupus erythematosus (adult epidermis).

Human herpesvirus 6 is present in lesions of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a disease characterized by Langerhans cell infiltration of skin and bone, with its most severe form manifested by multifocal infiltration of many organs. The etiology is unknown, although viral infection has been proposed as a potential pathogenic factor. Human herpesvirus 6 (HHV-6), a recently described member of the human herpesvirus family, has been associated with atypical or malignant lymphocytic processes, and immune disorders. Based on these observations, we suspected that HHV-6 may play a role in the pathogenesis of LCH. Lesional tissue of 30 patients with LCH was retrospectively examined for the presence of HHV-6 by using the polymerase chain reaction. Tissue specimens from 63 patients with other benign and malignant histiocytic and lymphocytic diseases served as controls. In addition, all specimens were examined with control primers specific for herpes simplex virus (HSV). HHV-6 DNA was detected in lesions of 14 of 30 patients with LCH (47%). On clinical subgroup analysis, HHV-6 DNA was found in 10 of 16 patients with extraosseous disease (63%) and in four of 14 patients with disease limited to bone (29%). In each case, the prevalence of HHV-6 in LCH lesions was statistically significant, when compared to the control population. HSV DNA was not found in any of the LCH or control specimens. Although the presence of a virus alone does not establish a causal role in the disease, it supports the possibility of an etiologic relationship. From this study, we emphasize the need for further investigation of the potential HHV-6-mediated pathogenesis of LCH.

Study of HLA-DR synthesis in cultured human keratinocytes.

Within the normal human epidermis only Langerhans and indeterminate cells express HLA-DR. Human keratinocytes (HK), however, may also express HLA-DR in certain disease states characterized by mononuclear cell infiltrates. Previous studies have shown that HK synthesize HLA-DR in response to stimulation by interferon gamma (INF-gamma). The purposes of this study were to define conditions under which cultured HK might express HLA-DR and to compare the HLA-DR synthesis of HK with that of monocytes. HLA-DR expression by HK as determined by indirect immunofluorescence of HK cultures was absent under standard low calcium conditions and remained absent with the addition of calcium, serum, mitogens, and supernatants from Pam-212 cells containing epidermal thymocyte-activating factor. HLA-DR expression in HK was induced by cocultivation with concanavalin A-stimulated peripheral blood mononuclear cells (PBMC), but not unstimulated PBMC. This effect was time-dependent and directly related to the number of PBMC. HLA-DR expression was also induced in a time- and dose-dependent manner by addition of supernatant from stimulated PBMC (SS) or by addition of recombinant INF-gamma but not by addition of interleukin (IL)-1 or IL-2. Induction by either SS or INF-gamma was blocked by an antiserum to INF-gamma. As determined by a semiquantitative immunoprecipitation technique, HLA-DR synthesis by HK was directly related to INF-gamma concentration. The pattern of HLA-DR peptides produced by HK was similar to that of monocytes, but the relative quantity synthesized was far less than that of monocytes.

Development of an in vitro keratinocyte model for use in the study of HSV specific cytotoxicity.

Herpes simplex virus (HSV) specific immune mediated cytotoxicity may be involved in control of HSV infections and in the tissue damage induced by HSV infection or HSV related skin disease such as herpes associated erythema multiforme. Developing an in vitro model to study this process has proved difficult due to the lack of an appropriate target cell that will express HSV antigens but is not simultaneously subject to viral induced cell death. The purpose of this study was to develop a model in which keratinocytes express cell surface HSV specific antigens but at the same time are protected from death due to viral infection. We found that keratinocytes infected with HSV in the presence of acyclovir (ACV) expressed such antigens yet remained viable for a period of time after the onset of antigen expression such that cytotoxicity studies could be successfully performed. Rabbit skin cells, a transformed keratinocyte line, or second passage human neonatal foreskin keratinocytes were grown in culture medium with or without 200 microM ACV and were infected with HSV. Examination by direct immunofluorescence with anti-HSV antibody revealed uniform HSV antigen expression by cells both with and without ACV by 8 h after infection. Cells infected without ACV exhibited marked structural abnormalities including formation of multinucleated giant cells, while cells with ACV showed fewer such changes throughout a 24-h period. An Ethidium Bromide-Acridine Orange cytotoxicity assay demonstrated significant increases in the cytotoxicity of infected cells not protected by ACV compared to that of cells with ACV (p less than .001). This in vitro model should prove useful in the investigation of HSV specific immune mediated cytotoxicity.

Cryoglobulinemia and decreased monocyte chemotaxis is malignant melanoma.

The sera fromm 13 patients with malignant melanoma were evaluated for immune complexes by cryoprecipitation and the 125I Clq binding assay. Cryoprecipitates were identified in 12/13 patients (92%) and cryoimmunoglobulins in 7/13 patients (54%). Either cryoimmunoglobulin or elevated Clq binding was identified in 8/13 patients (62%). Incubation of normal monocytes with the resuspended cryoimmunoglobulin of 7 melanoma patients produced greater than 50% reduction in the ability of th monocytes to respond to chemotactic stimuli (p < .01). Similar inhibition was seen with cryoimmunoglobulin from erythema multiforme patients, but not with 'medium alone, albumin, heat aggregated albumin or heat aggregated-IgG in similar concentrations. No soluble factors produced in vitro could be demonstrated to produce this inhibition. Inhibition of monocyte function by immune complexes may be an important component of impaired host response to malignant melanoma, or alternatively may represent an important mechanism for the accumulation of monocytes at sites of inflammation, analogous to migration inhibition factor.

IgA antibodies in chronic bullous disease of childhood react with 97 kDa basement membrane zone protein.

Chronic bullous disease of childhood (CBDC) is an autoimmune blistering disease occurring in prepubertal children. Both CBDC and its adult counter-part, linear IgA bullous dermatosis (LABD), are characterized by linear deposition of IgA along the cutaneous basement membrane zone (BMZ). Circulating IgA antibody in LABD has been found to bind to a 97-kDa BMZ antigen, whereas the antigen in CBDC has not been well characterized. The purpose of this study was to evaluate the immunoreactivity of BMZ IgA antibodies in a series of CBDC patients. We evaluated 12 sera from patients with CBDC with circulating IgA anti-BMZ antibodies on indirect immunofluorescence (IIF), which stained the epidermal side of split skin with titers ranging from 1:20 to 1:640. Immunoblotting was performed against two preparations of BMZ proteins: one enriched with the two bullous pemphigoid antigens (BP230, BP180) and one enriched with the LABD antigen (LABD97). Eight of the twelve sera reacted with a 97-kDa protein that co-migrated with the protein detected in many LABD sera. The intensity of the reaction on immunoblot correlated with serum antibody titers. There was no consistent pattern of reactivity of the IgA anti-BMZ antibodies with either the BP230 or BP180 antigens, although two sera reacted with several higher molecular mass proteins (160-200 kDa). The significance of this reactivity was examined with immunoblotting using BMZ-affinity-purified antibodies, and ELF using nitrocellulose-eluted antibodies. One serum also contained anti-BMZ IgA antibodies that reacted with a 180-kDa protein, corresponding to BP180. We conclude that IgA antibodies in CBDC sera recognize a 97-kDa BMZ antigen present on the epidermal side of BMZ split skin that co-migrates with the antigen previously identified in LABD. These findings suggest that CBDC and LABD are the immunologically related disorders occurring in different age groups.

Identification of functional platelet-activating factor receptors on human keratinocytes.

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Expression of fibronectin, laminin, and factor VIII-related antigen during development of the human cutaneous microvasculature.

The regulation of angiogenesis during human skin development is poorly understood. Since fibronectin is involved in cell movement and organization during embryogenesis and morphogenesis in a variety of species, we investigated the expression of fibronectin and factor VIII-related antigen, a marker for endothelial cells, at various stages in the development of the human cutaneous microvasculature. Skin specimens were obtained from 4 human fetuses during the second trimester (14-18 weeks), from newborn foreskins, and from consenting normal adults. Cryostat sections were stained with both fluorescein-conjugated rabbit antihuman fibronectin and rhodamine-conjugated goat antihuman factor VIII-related antigen. Expression of fibronectin in the microvasculature was striking in fetal skin but became progressively less prominent with maturation. Fibronectin appeared in fetal blood vessels as a bright continuous linear array, in neonatal blood vessels as a bright interrupted linear and speckled array, and in adult blood vessels as a sparse interrupted linear and speckled array. In contrast, expression of factor VIII-related antigen by the endothelium became more prominent with the degree of maturation of the microvasculature. Granular factor VIII-related antigen staining was scant in the newly forming blood vessels of fetal skin, bright but focal in the microvasculature of newborn skin, and intense and almost confluent in the blood vessels of adult skin. Although expression of fibronectin and factor VIII-related antigen changed, expression of laminin was consistent throughout development. Staining of the same skin specimens with fluorescein-conjugated sheep antihuman laminin produced a bright continuous linear pattern in all blood vessels. The reciprocal relationship manifested by intense fibronectin staining during human blood vessel development and prominent factor VIII-related antigen staining in mature blood vessels supports the hypotheses that fibronectin plays a role in human blood vessel modulation and morphogenesis, and that factor VIII-related antigen is a marker for endothelial cell differentiation.