Biomedical subjects
B A Gilchrest
Publications and source records attributed to B A Gilchrest.
Growth factor responsiveness declines during adulthood for human skin-derived cells.
Keratinocytes and fibroblasts were obtained from upper arm biopsies of young (22-27 years) and old (60-82 years) adult donors. Keratinocytes were grown in serum-free medium containing variable quantities of either epidermal growth factor (EGF) or a bovine hypothalamic extract known to contain keratinocyte growth factor (KGF). Fibroblasts were grown in serum-free medium containing variable quantities of EGF or insulin. Paired keratinocyte cultures were plated in serum-free medium containing 20% newborn keratinocyte-conditioned medium (NM) or 20% control conditioned medium (CM). Newborn foreskin keratinocytes were plated in 20% conditioned media derived from newborn, young adult or old adult keratinocyte cultures. In spite of large inter-donor variability, keratinocyte growth significantly decreased with age (0.05 greater than P greater than 0.01). Cell yield at 7 days showed an 8-fold increase for young adults over the KGF dose range treated, but only a 4-fold increase for old adults. Young adult cells in varying concentrations of EGF achieved 3-fold to 5-fold higher densities than old, although EGF was not stimulatory for either adult age group. Donor age-associated loss of growth factor responsiveness was confirmed with dermal fibroblasts derived from the same biopsies. Newborn but not adult keratinocytes were stimulated by NM, while newborn cells were not stimulated by either young or old adult conditioned media (YM or OM). An epidermal proliferation index, incorporating both donor cell yield and cell yield of newborn cells in donor conditioned medium, was significantly different (P less than 0.01) for newborn vs. young or old adult cells. Our findings confirm that a decreased proliferative capacity is measurable within adulthood, and suggest that this decrease may be due to a reduced ability to synthesize or respond to mitogens, including autocrine factors.
Normal human epidermis contains an interferon-like protein.
Interferons have been postulated to participate in growth regulation of normal body tissues and are known to inhibit growth of human epidermal keratinocytes in vitro. Polyclonal antibodies to recombinant human interferon-alpha, purified by passage over an affinity column (Sepharose coupled to the recombinant interferon), used in the indirect immunofluorescent method specifically stained the proliferative (basal) compartment of human epidermis in histological cross-sections of normal skin and in cultured keratinocyte colonies. Extracts prepared from healthy nonvirally infected keratinocyte cultures contained interferon activity as determined by viral plaque inhibition assay. Using the Western blotting technique column-purified antibodies and antisera to recombinant human interferon-alpha recognized a band of approximately 40 kD when reacted with both extracted keratinocyte proteins and recombinant human interferon-alpha standards, that gave in addition a band of approximately 20 kD. The above findings suggest that interferon or a closely related protein is present in the proliferative compartment of normal epidermis in the absence of viral infection and therefore may serve as a physiological modulator of epidermal growth.
Relative responsiveness of cultured human epidermal melanocytes and melanoma cells to selected mitogens.
Early cellular events in the malignant transformation of melanocytes to melanoma are virtually unknown. In vitro investigation of this phenomenon has been hampered by the fastidious nature of the human epidermal melanocyte, which has proven difficult to cultivate. The present study compares responsiveness of cultured human epidermal melanocytes and established melanoma cell lines to serum, cholera toxin, and melanocyte growth factor (MGF), three established melanocyte mitogens. Four of four established human melanoma lines were substantially stimulated by fetal bovine serum, as were newborn foreskin-derived epidermal melanocytes. In contrast, none of the four melanoma lines responded to hypothalamic preparations containing MGF that consistently produced an approximately 30-fold increase in newborn melanocyte cell yield over a 2-week period. Cholera toxin, required for successful establishment of primary melanocyte cultures, had small and variable effects on the melanoma lines, with slight stimulation in one case, moderate inhibition in another, and essentially no effect in two others. These data suggest that transformation of epidermal melanocytes to melanoma often involves at least one phenotypic change resulting in escape from MGF regulation and another associated with insensitivity to cyclic AMP modulation; while at least some of the pathways conferring serum dependence are unaltered. Improved culture systems for the human epidermal melanocyte should facilitate further studies into the mechanism of its malignant conversion and may provide useful insights for the prevention and treatment of human melanoma.
Characteristics of cultivated adult human nevocellular nevus cells.
Nevus cells are of biologic interest because of their uncertain relationship to epidermal melanocytes and of clinical interest because of their statistical association with melanoma. We report a technique that allows reliable cultivation of nevus cells from small acquired and congenital nevi and permits in vitro characterization of this cell type. Morphologically, cultured nevus cells were found to closely resemble epidermal melanocytes from the same or comparably aged donors, manifesting marked dendricity and specific ultrastructural features characteristic of melanocytes; but could be distinguished by the presence of occasional large binucleate or trinucleate cells and by the frequent finding of grouped melanosomes in nevus cell cytoplasm. Growth kinetics were also similar for nevus cells and epidermal melanocytes, with population doubling times of 1-2 weeks in hormone-supplemented serum-free medium, and substantial growth enhancement by fetal bovine serum. As previously noted for epidermal melanocytes, nevus cells in serum-free culture demonstrated striking substrate responsiveness, with far greater attachment rates and degree of cytoplasmic spreading on fibronectin or type I/III collagen than on laminin, type IV collagen, or uncoated plastic. These strong similarities in vitro suggest that morphologic and behavioral differences observed between epidermal melanocytes and nevus cells in the skin may result from local environmental influences rather than from intrinsic cellular differences. The availability of a satisfactory culture system for nevus cells may facilitate future investigations into their malignant potential and other biologic features.
Defined culture systems for pharmacological and toxicological studies.
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Influence of increased extracellular calcium concentration and donor age on density-dependent growth inhibition of human fibroblasts.
Elevated calcium chloride concentration [( CaCl2]) has been shown to increase saturation density for an established mouse fibroblast line and for human fetal lung fibroblasts (WI-38). In order to examine the effect of increased [CaCl2] on human fibroblasts from donors of varying age, fibroblasts were grown in medium (basal level of 1.8 mM CaCl2) supplemented with fetal bovine serum (FBS) until confluent. Compared to controls in basal medium, newborn foreskin fibroblasts exposed to additional CaCl2 had a 110-450% increased cell yield that was independent of [CaCl2] within the range of an additional 1.5-5.0 mM. The effect was maintained over an eightfold range of FBS concentration. Initial growth rate was unaffected, but a prolongation of exponential phase occurred for cultures exposed to increased [CaCl2]. Confluent cultures refed medium with increased [CaCl2] were stimulated 5- to 10-fold more than cultures refed basal medium. An additional 2 mM CaCl2 resulted in a 210% increase for young adult-derived fibroblasts versus a 29% increase for old adult-derived fibroblasts (P less than 0.001). These data indicate that increased [CaCl2] decreases density-dependent growth inhibition of postnatal human dermal fibroblasts in vitro and that this effect is donor age dependent.
The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain.
Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.
Substrate influences human epidermal melanocyte attachment and spreading in vitro.
Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristic melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo.
Effects of alpha and beta interferons on cultured human keratinocytes.
Interferons (IF) are a family of glycoproteins known for their antiviral activity and the ability to inhibit growth and alter behavior of various normal and transformed cell types, both in vivo and in vitro. Because cultured human keratinocytes (HK) produce IF in response to viral infection, we undertook studies of alpha-IF and beta-IF effects on HK. Cloned human IF were added at time of seeding and at each feeding to paired dishes of keratinocytes maintained in serum-free hormone-supplemented medium. At 7 days significant inhibition of growth was observed for both alpha-IF and beta-IF, as determined by cell counts, total protein, and appearance of stained colonies, and was sustained for at least two weeks during continuous IF exposure. The inhibition was substantially blocked by prior addition of cholera toxin to the medium, consistent with competition for a common cell surface receptor. Growth of a single human epidermal carcinoma cell line was much less affected by IF than was growth of the normal keratinocytes. IF also promoted terminal differentiation of keratinocytes as assessed by desquamation rate of cells from the colony surface and by proportion of total cells having cornified envelopes. IF effect on both growth and differentiation was completely reversible within days of its removal from medium. These findings suggest that IF may function as a physiologic regulator of epidermal growth in vivo with properties of a negative growth factor or chalone.
Characterization and partial purification of keratinocyte growth factor from the hypothalamus.
An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.
Pruritus, cutaneous pain, and eccrine gland and sweating disorders.
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Virus replication and induction of interferon in human epidermal keratinocytes following infection with herpes simplex virus.
Keratinocyte cultures derived from surgical skin specimens of healthy newborns and adults were infected with herpes simplex virus (HSV) type 1 or 2. Typical HSV cytopathic effects involved all cell layers in stratified colonies, and paralleled the production of infectious virus. Virus growth curves and production of virus were comparable in newborn and adult keratinocytes. Interferon (IF) production by keratinocytes paralleled the yield of virus over at least 72 h, and was greater in cultures of adult cells than cultures from newborns. UV irradiation of HSV resulted in progressive virus inactivation and a parallel reduction in induced IF. This suggests that IF production was related to virus replication, and that irradiated (noninfectious) HSV DNA did not contribute significantly to the generation of IF in this system. These results establish that human epidermal keratinocytes can serve as a model system for quantitative assessment of herpes simplex virus infection.
Selective cultivation of human melanocytes from newborn and adult epidermis.
Development of adequate culture systems for the human epidermal melanocyte is critical to further advances in pigment cell biology. We now report selective growth and long-term maintenance of melanocytes derived from both newborn and adult skin specimens. Disaggregated epidermal cell suspensions were plated in a hormone-supplemented medium containing cholera toxin and a hypothalamic extract treated to remove keratinocyte growth-promoting activity. After 3-4 weeks, pure melanocyte populations could be harvested and serially passaged up to 6 times over several months for a total of 10 or more cumulative population doublings in vitro. Electron microscopic studies revealed metabolically active cells with abundant melanosomes in various stages of melanization throughout the culture lifespan. Differences in size and number of melanosomes attributable to race of the tissue donor were readily apparent, and pigment content of melanocytes from both black and Caucasian donors appeared to increase with time in culture. Newborn melanocytes proliferated more rapidly and survived longer than did adult melanocytes, but there were no consistent morphologic differences as a function of donor age. Comparison of growth potential for the 3 major skin-derived cell types in this hormone-supplemented medium revealed striking specificity for melanocytes, with total elimination of keratinocytes over 1-2 weeks, and no fibroblast proliferation whatever in the absence of serum supplementation. This system promises to facilitate in vitro investigation of epidermal melanocytes in normal and diseased human skin.
Autocrine and paracrine growth stimulation of cells derived from human skin.
A sensitive serum-free culture system was used to demonstrate that cells derived from normal human skin release soluble mediators which can modulate keratinocyte, melanocyte, and fibroblast growth in vitro. In M199 supplemented with epidermal growth factor, hydrocortisone, insulin, transferrin, triidothyronine, bovine serum albumin, and an extract of bovine hypothalamus, keratinocytes underwent approximately three to five cumulative population doublings (CPD) over a 7-day period. Addition of 20% keratinocyte-conditioned medium more than doubled keratinocyte growth and increased fibroblast growth 25-40% above controls. Dermal fibroblasts maintained in the same serum-free hormone-supplemented medium (SFHSM) alone underwent approximately 4.5-5.5 CPD over a 7-day period; 20% fibroblast-conditioned medium increased fibroblast growth 50-80% and increased keratinocyte growth 30-50%. Melanocytes maintained in the same SFHSM underwent approximately 1 CPD over a 14-day period and 2 CPD if the medium was supplemented with 2% fetal bovine serum (FBS). Addition of 20% melanocyte-conditioned medium more than doubled melanocyte growth in either SFHSM or in medium containing 2% FBS, but decreased both keratinocyte and fibroblast growth up to 30-60%. None of the conditioned media altered cellular morphology. These data provide the first demonstration of mutual growth modulation by cell types normally contiguous in vivo and expand existing evidence for autocrine and paracrine growth regulation by normal human cells.
Histologic changes associated with ultraviolet A--induced erythema in normal human skin.
We have examined the effects of a standardized, moderately erythemogenic dose of long-wave ultraviolet (UVA) radiation on normal human skin, with the use of an appropriately filtered solar simulator and sequential biopsy specimens processed as 1-micron Epon-embedded sections. Histologic changes were present immediately after irradiation and evolved slowly during the 48-hour study. The epidermis manifested slight intracellular and intercellular edema and progressive loss of Langerhans cells to approximately one-fifth control values. A dermal infiltrate of neutrophilic polymorphonuclear leukocytes was present in all postirradiation specimens and peaked at 3 hours. A perivascular lymphocytic infiltrate, moderate endothelial cell enlargement, mast cell hypogranulation, occasional massive venular dilation, and sparse red blood cell extravasation were also noted. Overall, our findings expand and quantify earlier impressions that, compared to UVB, UVA has a relatively greater histologic effect on the dermis than on the epidermis, depletes epidermal Langerhans cells, and recruits neutrophils into irradiated human skin.
Growth factor responsiveness of cultured human fibroblasts declines with age.
A sensitive culture system was used to investigate whether in vitro senescence is associated with loss of responsiveness to relevant growth factors in the cellular environment. Early passage dermal fibroblasts obtained from healthy newborns and adults were maintained 4 to 6 days in medium containing either fetal calf serum or five defined growth factors. Newborn fibroblasts consistently grew far better than adult fibroblasts in both media; dose-response curves demonstrated a greater response by newborn fibroblasts to both serum mitogens and to thrombin, insulin, and epidermal growth factor alone and in combination. These data suggest that newborn fibroblasts generally have greater mitogenic responsiveness than adult fibroblasts and that age-associated loss of growth factor responsiveness may indeed contribute to the decreased proliferative capacity of old cells.