Joel S. Trupin

Chemokine and chemokine receptor expression in keloid and normal fibroblasts

Keloids are benign collagenous tumors that occur during dermal wound healing in genetically predisposed individuals. The lesions are characterized by over‐proliferation of fibroblasts, some leukocyte infiltration, and prolonged high rates of collagen synthesis. To determine whether leukocyte chemoattractants or chemokines are participating in this disease process, immunohistochemical staining for the CXC chemokine, MGSA/GROα, and its receptor, CXCR2, was performed on tissue from keloids, hypertrophic scars and normal skin. Immunoreactive MGSA/GROα was not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts and lymphocytes in nodular areas of the keloid samples. This staining positively correlated with the degree of inflammatory infiltrate in the lesions. Keloids, but not hypertrophic scars or normal dermis, also exhibited intensive immunoreactivity for the CXCR2 receptor in endothelial cells and inflammatory infiltrates with occasional staining of myofibroblasts. In contrast, cultured fibroblasts from either keloids or normal skin did not express detectable amounts of mRNA for MGSA/GRO or CXCR2, although interleukin‐1 strongly induced MGSA/GRO mRNA in both cell types. Interleukin‐1 induction of MGSA/GRO was inhibited by glucocorticoid in normal and keloid fibroblasts, and the effect was more pronounced in keloid fibroblasts. This event was not correlated with inhibition of nuclear activation of NF‐κB, AP‐1 or Sp1, and might therefore be mediated by another mechanism such as decreased mRNA stability or transcriptional repression through the glucocorticoid response element in the MGSA/GRO promoter. Data from in vitro wounding experiments with cultured normal and keloid fibroblasts indicate that there were no significant differences in MGSA/GRO or CXCR2 receptor levels between normal and keloid fibroblasts. We also show that cultured keloid fibroblasts exhibit a delayed wound healing response. We postulate that the inflammatory component is important in development of keloid lesions and chemotactic cytokines may participate in this process.

Variation in Prolyl Hydroxylase Activity of Keloid-Derived and Normal Human Fibroblasts in Response to Hydrocortisone and Ascorbic Acid

The effects of ascorbic acid and hydrocortisone on activity of prolyl hydroxylase in fibroblasts from keloid and normal human dermis were investigated and compared to the effects of these agents on collagen synthesis. Prolyl hydroxylase activity in normal fibroblasts grown to confluency in 1.5 microM hydrocortisone was approximately half that of cells grown without the steroid. The concentration of hydrocortisone effective in reducing enzyme activity was the same as that for reducing the rate of collagen synthesis; a half-maximal effect on both parameters was achieved at 10(-7) M. Hydrocortisone lowered enzyme activity through most of the culture cycle. Fibroblasts derived from keloids were significantly less subject to hydrocortisone-mediated reduction of prolyl hydroxylase activity and rate of collagen synthesis. This difference between keloid and normal cells was dependent on the simultaneous presence of ascorbic acid and hydrocortisone. These data suggest that the defect in wound healing that results in keloid formation is associated with a change in a regulatory mechanism that controls the rate of collagen synthesis and is sensitive to physiological levels of hydrocortisone. Continuous culture of fibroblasts in medium supplemented with ascorbic acid also lowered prolyl hydroxylase activity. Unlike the effect of hydrocortisone, growth in ascorbate increased the rate of collagen synthesis and affected keloid and normal strains equally.

Pleiotropic Effects of Immune Responses Explain Variation in the Prevalence of Fibroproliferative Diseases.

Many diseases are differentially distributed among human populations. Differential selection on genetic variants in ancestral environments that coincidentally predispose to disease can be an underlying cause of these unequal prevalence patterns. Selected genes may be pleiotropic, affecting multiple phenotypes and resulting in more than one disease or trait. Patterns of pleiotropy may be helpful in understanding the underlying causes of an array of conditions in a population. For example, several fibroproliferative diseases are more prevalent and severe in populations of sub-Saharan ancestry. We propose that this disparity is due to selection for an enhanced Th2 response that confers resistance to helminthic infections, and concurrently increases susceptibility to fibrosis due to the profibrotic action of Th2 cytokines. Many studies on selection of Th2-related genes for host resistance to helminths have been reported, but the pleiotropic impact of this selection on the distribution of fibrotic disorders has not been explicitly investigated. We discuss the disproportionate occurrence of fibroproliferative diseases in individuals of African ancestry and provide evidence that adaptation of the immune system has shaped the genetic structure of these human populations in ways that alter the distribution of multiple fibroproliferative diseases.

Assay of prolyl hydroxylase in cultured fibroblast monolayers

Abstract Prolyl hydroxylase may be assayed in cultured cell monolayers after a cold acetone treatment or air-drying. Enzyme activity of dried cells is stable when cell monolayers are stored frozen and desiccated, thus permitting simultaneous assay of cells harvested at different times, e.g., on different days of a culture cycle. Uniformity of cell monolayers permits use of separate dishes for assay of protein or DNA to determine specific enzyme activity. This procedure greatly facilitates harvesting and assay of cells for a variety of biochemical measurements.