In situ changes in the relative abundance of human epidermal cytokine messenger RNA levels following exposure to the poison ivy/oak contact allergen urushiol.

Abstract: Epidermal keratinocytes in culture have been shown to produce many cytokines, and their proteins have been identified in skin tissue samples. It has therefore been assumed that these cytokines are transcribed in vivo by the epidermis in response to contact allergens. In this report, in situ hybridization was used to detect the messenger RNAs for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) in samples of human skin prior to and at various times after application of urushiol, the immunogenic component of poison ivy/oak. In sensitive subjects, IL-1 alpha and TNF-alpha mRNAs showed a progressive increase in transcript levels that paralleled the clinical and histological features of the inflammatory process. The time-course of the IL-1 beta response differed from that of IL-1 alpha and TNF-alpha, in that there was an early (by 6 h after urushiol administration) elevation in IL-1 beta mRNA that occurred before there was evidence of inflammation and had returned to background levels by 72 h when the reaction had reached its peak. In contrast to urushiol-sensitive subjects, urushiol-anergic individuals did not exhibit an increase in IL-1 alpha, IL-1 beta or TNF-alpha mRNA levels. The data provide evidence for an in vivo role for epidermal IL-1 alpha, IL-1 beta and TNF-alpha transcription in the regulation of IL-1 beta and TNF-alpha polypeptide levels in the epidermis in response to this common contact allergen.

Role of tumor necrosis factor and granulocyte-macrophage colony-stimulating factor in the late allergic response in human nasal mucosa.

Cytokines play an integral role in the allergic response of the nasal mucosa. The ideal model for analysis of this interaction has yet to be perfected. We present a model for such evaluation and present results of experiments on the release of several cytokines. Freshly harvested human nasal turbinate mucosa was placed on a Gelfoam (Upjohn Co., Kalamazoo, Mich.) raft in a liquid medium to simulate the in situ environment. The allergic response was initiated by exposing the nasal mucosa to various combinations and amounts of human immunoglobulin E and antihuman immunoglobulin E antibody. The supernatants were collected and analyzed by enzyme-linked immunosorbent assay techniques for various cytokines. Histopathologic evaluation of the mucosa was performed throughout the exposure period, confirming normal cellular and tissue architecture and viability. This model was used to monitor the release of interleukin-3, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptors after exposure to immunoglobulin E and immunoglobulin E antibody. Interleukin-3 did not show significant increases during the experiment testing period of 48 hours. Tumor necrosis factor-alpha and soluble tumor necrosis factor receptors demonstrated time-dependent increases in concentration after immunoglobulin E stimulation. Granulocyte-macrophage colony-stimulating factor showed the greatest time-dependent increases. Their impact on the understanding of the allergic response will be discussed.

Messenger RNAs for the multifunctional cytokines interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha are present in adnexal tissues and in dermis of normal human skin.

Interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are 3 cytokines that play a key rôle in cutaneous homeostasis and in the immunopathogenesis of a number of dermatologic diseases. Most studies have focused on their production by keratinocytes and Langerhans cells. To determine whether there are non-epidermal sites of cytokine transcription, biopsy specimens of normal human skin were probed for IL-1 alpha, IL-1 beta and TNF-alpha messenger RNAs using the method of in situ hybridization. The results demonstrate that each cytokine mRNA is present at multiple sites within the skin, including epidermal appendages and adnexal structures (hair follicles, sebaceous glands), the dermal microvasculature, arrectores pilorum smooth muscle, and the dermal connective tissue. These data provide evidence that in vivo there are multiple sites other than the epidermis of constitutive IL-1 alpha, IL-1 beta, and TNF-alpha gene transcription in normal human skin.

Insulin levels, blood pressure and sleep apnea.

This report concerns the relative contributions of body weight and sleep apnea to the following cardiovascular risk factors: blood pressure, fasting insulin and fasting glucose. We cross-sectionally examined the relationship of various levels of apneic activity [apnea-hypopnea index (AHI)] and a measure of obesity [body mass index (BMI)] to mean morning blood pressure and fasting serum insulin and fasting blood glucose concentrations sampled the morning after polysomnography. Subjects were 261 males (age 47 +/- 13 years, mean +/- SD), who were referred to a sleep laboratory for symptoms of sleep-disordered breathing. The dependent variables, mean morning blood pressure, insulin and fasting blood glucose (FBG) levels, were significantly related to both AHI (eta'2 = 0.10) and BMI (eta'2 = 0.18). AHI and BMI combined to account for approximately 30% of the variability in the best linear combination of these three factors. Further analysis indicated that mean morning blood pressure and fasting insulin levels each correlated positively with BMI and AHI, whereas FBG correlated only with BMI. We conclude that, although these data do not prove a causal relationship, there is evidence for an independent association between sleep apnea and not only blood pressure, but also fasting insulin levels.

In situ detection of cytokine messenger RNAs in the eccrine sweat gland of normal human skin.

Human sweat and eccrine sweat glands contained the multifunctional polypeptide cytokines, interleukin-1 alpha and beta (IL-1 alpha and beta). To determine whether the sweat gland itself is an actual site of transcription for cytokines we performed in situ hybridization on histologic sections of normal human skin. In this report we show that the mRNAs encoding the cytokines IL-1 alpha, IL-1 beta, and tumor necrosis factor-alpha (TNF-alpha) are present in normal human skin eccrine sweat gland duct and secretory coil epithelium. These results suggest that in vivo the sweat gland is a production site for cytokine polypeptides and may contribute to the exceedingly large quantities of these cytokine proteins found in the epidermis.

Cellular localization and regional distribution of an angiotensin II-forming chymase in the heart.

The human heart is a target organ for the octapeptide hormone, angiotensin II (Ang II). Recent studies suggest that the human heart contains a dual pathway of Ang II formation in which the major Ang II-forming enzymes are angiotensin I-converting enzyme (ACE) and chymase. Human heart chymase has recently been purified and its cDNA and gene cloned. This cardiac serine proteinase is the most efficient and specific Ang II-forming enzyme described. To obtain insights into the cardiac sites of chymase-dependent Ang II formation, we examined the cellular localization and regional distribution of chymase in the human heart. Electron microscope immunocytochemistry using an anti-human chymase antibody showed the presence of chymase-like immunoreactivity in the cardiac interstitium and in cytosolic granules of mast cells, endothelial cells, and some mesenchymal interstitial cells. In the cardiac interstitium, chymase-like immunoreactivity is associated with the extracellular matrix. In situ hybridization studies further indicated that chymase mRNA is expressed in endothelial cells and in interstitial cells, including mast cells. Tissue chymase levels were determined by activity assays and by Western blot analyses. Chymase levels were approximately twofold higher in ventricles than in atria. There were no significant differences in chymase levels in ventricular tissues obtained from non-failing donor hearts, failing ischemic hearts, or hearts from patients with ischemic cardiomyopathy. These findings suggest that a major site of chymase-dependent Ang II formation in the heart is the interstitium and that cardiac mast cells, mesenchymal interstitial cells, and endothelial cells are the cellular sites of synthesis and storage of chymase. In the human heart, because ACE levels are highest in the atria and chymase levels are highest in ventricles, it is likely that the relative contribution of ACE and chymase to cardiac Ang II formation varies with the cardiac chamber. Such differences may lead to differential suppression of cardiac Ang II levels during chronic ACE inhibitor therapy in patients with congestive heart failure.

Modulation of interferon-gamma-induced HLA-DR expression on the human keratinocyte cell line SCC-13 by ultraviolet radiation.

Cell surface expression of major histocompatibility determinants on epidermal keratinocytes is a characteristic feature of a number of inflammatory dermatoses and in all likelihood is caused by diffusion of human leukocyte antigen (HLA)-DR-inducing cytokines from cells present in the dermal mononuclear cell infiltrate. Many of these same disorders respond to ultraviolet (UV) radiation phototherapy. Using the human SCC-13 keratinocyte cell line as a model, UV radiation was found to inhibit interferon-gamma-induced HLA-DR expression. Inhibition correlated closely with decreased steady-state levels of HLA-DR mRNA. These findings provide evidence that the therapeutic effect of UV radiation phototherapy may be mediated by its capacity to down-regulate cytokine-induced keratinocyte HLA-DR expression.

Biochemical morbidity in sleep apnea.

The long-term goals of our research are to understand the biochemical morbidity surrounding obstructive sleep apnea syndrome to define better the need for treatment and to determine modifiable risk factors for the disease. Our current hypothesis is that sleep-related hypoxemia results in alterations in metabolic regulatory peptides, specifically insulin and insulin-like growth factors (IGF-1 and IGF-2), which are known or suspected factors for obesity and disorders such as hypertension, glucose intolerance, and atherosclerosis. Surveys of clinic populations suggest a relationship between body habitus, parameters of sleep-disordered breathing, indices of oxygenation, and insulin resistance, defined by fasting serum levels of glucose and insulin. Results will provide insight into the role of metabolic regulatory peptides in the pathogenesis of sleep-disordered breathing and the mechanisms for this association.

Cryptococcal osteomyelitis and cellular immunodeficiency associated with interleukin-2 deficiency.

We describe an unusual example of cellular immunodeficiency associated with interleukin-2 deficiency in an otherwise healthy 15-year-old boy who had isolated cryptococcal osteomyelitis of the scapula at 10 years of age. His previous medical history was remarkable only for prolonged, severe varicella infection at 6 years of age. He had persistent moderate lymphopenia, anergy, and absent lymphocyte blastogenic responses to mitogens, antigens, or monoclonal T cell antibodies. Subnormal blastogenic responses were seen after exposure to high concentrations of phorbol esters. Immunoglobulin levels and specific antibodies were normal. The patient has been in good health since treatment of his osteomyelitis. However, his lymphocyte blastogenic responses to mitogens have remained absent during 4 years of observation; investigation of the cause revealed a specific interleukin-2 deficiency resulting from defective generation of interleukin-2 messenger ribonucleic acid. Secretion of interleukin-1 by monocytes was normal, suggesting that the abnormal blastogenic response and interleukin-2 production were due to a problem intrinsic to T lymphocytes. The generation of messenger ribonucleic acid for interleukin-4 was not affected. Interferon-gamma was produced at subnormal levels. The addition of recombinant interleukin-2 restored lymphocyte blastogenic responses and increased the expression of interleukin-2 receptors. The clinical findings and immunologic abnormalities present in this patient differ from other primary and secondary immunodeficiencies associated with interleukin-2 deficiency. Thus our observations in this patient extend the spectrum of immunodeficiencies associated with abnormalities in the production of this important cytokine.

Transforming growth factor-beta 1 in heart development.

Defined biochemical stimuli regulating neonatal ventricular myocyte (cardiomyocyte) development have not been established. Since cardiomyocytes stop proliferating during the first 3-5 days of age in the rodent, locally generated 'anti-proliferative' and/or differentiation signals can be hypothesized. The transforming growth factor-beta (TGF-beta) family of peptides are multifunctional regulators of proliferation and differentiation of many different cell types. We have determined in neonatal and maturing rat hearts that TGF-beta 1 gene expression occurs in pups of both normotensive (Wistar Kyoto, WKY) and hypertrophy-prone rats (spontaneously hypertensive, SHR). TGF-beta 1 transcript levels were readily apparent in total ventricular RNA from SHR pups within 1 day of age and elevated in 3-7 day old WKY and SHR hearts when cardiomyocyte proliferation indices are diminished. TGF-beta 1 transcript levels remain at a 'relatively' high level throughout maturation and into adulthood in both strains. Further, TGF-beta 1 transcripts were localized to cardiomyocytes of neonatal rat ventricular tissue sections by in situ hybridization. Immunoreactive TGF-beta was co-localized to the intracellular compartment of neonatal cardiomyocytes at the light and electron microscopic level. In vitro analysis using primary cultures of fetal and neonatal cardiomyocytes indicated that TGF-beta s inhibit mitogen stimulated DNA synthesis and thymidine incorporation. From these data, we propose that locally generated TGF-beta s may act as autocrine and/or paracrine regulators of cardiomyocyte proliferation and differentiation as intrinsic components of a multifaceted biochemical regulatory process governing heart development.

Expression of the insulin-like and platelet-derived growth factor genes in human uterine tissues.

The human uterus repeatedly exhibits cyclic biochemical and cytological changes during the reproductive period of life. These changes are the result of a well-characterized endocrine network involving the hypothalamus, pituitary, and ovary. The exact nature of the mechanism(s) by which the sex steroids act on the uterus remains to be elucidated. Possible local mediators of hormonal action on the uterus include polypeptide growth factors. Using the method of RNA transfer blot hybridization, we have analyzed tissue samples from the cycling human endometrium and tissue samples of human myometrium and myometrial benign tumor (leiomyoma) for the presence of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) RNA. All the uterine tissues examined possessed RNA for PDGF-B chain and IGF-I and -II. Two transcripts were observed for PDGF-B chain, four were observed for IGF-I, and eight were observed for IGF-II. Overall, the relative abundance of PDGF-B chain RNA was consistent in all of the uterine tissues examined. In contrast, IGF RNA relative abundance varied. IGF-I RNA was highest in late proliferative stage endometrium, and IGF-II RNA was highest in early proliferative stage endometrium. Both IGF-I and IGF-II RNAs were greater in amount of leiomyoma than in myometrium. The increased IGF-I RNA in late proliferative-stage human endometrium correlates with the known elevation of estradiol secretion by the ovary and the increased concentration of uterine estradiol receptors during this stage of the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factors and neonatal cardiomyocyte development: ventricular gene expression and membrane receptor variations in normotensive and hypertensive rats.

Defined factors regulating or influencing mammalian ventricular myocyte (cardiomyocyte) development are not known at this time. During early neonatal ventricular growth, cardiomyocytes begin a 'transition phase' of development toward cellular maturation (hypertrophy) that entails terminal proliferation and cellular binucleation. Insulin-like growth factor-I and -II (IGFs) are believed to play a major role in mammalian postnatal and fetal growth, possibly functioning in local environments which facilitate autocrine or paracrine tissue growth characteristics. Therefore, we examined the expression of the IGF genes and their corresponding membrane receptors in ventricles of normotensive and spontaneously hypertensive (SHR) rat pups during the first 7-14 days of age. We have determined: (1) by receptor crosslinking that neonatal ventricular membranes possess type 1 and type 2 IGF receptors; (2) by receptor binding analysis that type 1 IGF receptor concentration is elevated between days 1-7 in the SHR and shows an age-related decline in concentration and an increase in affinity in both strains; (3) by Northern blot analysis that neonatal rat ventricular tissue expresses primarily IGF-II RNA transcripts of 3.6, 2.3 and 1.7 kilobases (kb) in size, with low levels of IGF-I transcripts detected; (4) by slot-blot hybridization that SHR ventricles contain higher levels of IGF-II transcripts at 3 days of age; and (5) localized the IGF transcripts to ventricular myocytes by tissue in situ hybridization. These observations support a role for cardiomyocyte-produced IGFs that may be locally produced and act in an autocrine or paracrine fashion to modulate cardiomyocyte growth and maturation in the developing rat heart. Because both IGF receptor and IGF RNA transcript parameters differed in SHR hearts, genetically predisposed to hypertrophy, a potentially important biochemical alteration may be associated with the fetal/neonatal growth abnormalities of the developing heart in this rat strain.

The interleukin 2 gene is expressed in the syncytiotrophoblast of the human placenta.

The lymphokine interleukin 2 is an important immune system regulatory glycopolypeptide. It is produced by antigen- or mitogen-stimulated T lymphocytes and is required for the proliferation or clonal expansion of activated T lymphocytes. In this report, it is demonstrated by RNA transfer blot hybridization that the poly(A)+ RNA population of the human placenta contains a 0.85-kilobase RNA transcript that specifically hybridizes to a human interleukin 2 cDNA probe. By using hybridization histochemistry in situ, it is further shown that interleukin 2 RNA transcripts are localized, primarily, to the syncytial (syncytiotrophoblast) layer of the human placenta. Possible roles for syncytiotrophoblast-produced interleukin 2 are suggested and discussed.

Induction of vitellogenin in primary monolayer cultures of cockerel hepatocytes.

A primary monolayer culture system from cockerel hepatocytes was established. The cultures synthesize and secrete proteins that comigrate with authentic serum proteins on polyacrylamide gels and are found in the same relative abundance. Addition of estradiol increased the synthesis of apoprotein B, found in very low density lipoprotein, under all culture conditions. Vitellogenin synthesis could not be induced directly by estradiol. However, when serum was obtained from cockerels injected with estradiol 4 days before blood collection and included in the culture medium, the cultures secreted a protein identified immunologically as vitellogenin by affinity chromatography. Furthermore, addition of growth hormone or prolactin to cultured cockerel hepatocyte monolayers resulted in the synthesis and secretion of a polypeptide that comigrates with authentic vitellogenin on polyacrylamide gels.