Wound Macrophages Express TGF-α and Other Growth Factors in Vivo: Analysis by mRNA Phenotyping

Abstract

The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.

Localization of Fas and Fas ligand in bone marrow cells demonstrating myelodysplasia

“…The PCR reaction was performed as described. 9,14,15 As a control reaction of PCR, ␤-actin was also used in each run. Briefly, 250 ng of the RNA was used for RT-PCR.…”

“…6 Hematopoietic cells as well as stromal cells have been reported to express a wide range of substances which may play complex regulatory roles in hematopoiesis. [7][8][9] We have reported previously that the number of macrophage lineage cells significantly increased in the bone marrow of MDS patients as compared with that of the control or de novo AML bone marrow. 10 Furthermore, bone marrow macrophages express tumor necrosis factor (TNF)␣ and interferon (IFN)-␥ which have an accelerating effect on apoptosis.…”

Overexpression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by bone marrow cells from patients with myelodysplastic syndromes

“…The PCR reaction was performed as described elsewhere. 21,23,24 As a control reaction of PCR, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (196 bp) was also detected in each run. Briefly, 50 ng of the RNA was used for RT-PCR.…”

“…18 Hematopoietic cells as well as stromal cells have been reported to express a wide range of substances which may play complex regulatory roles in hematopoiesis. [19][20][21] We have reported previously that the number of macrophage-lineage cells significantly increased in the bone marrow of MDS patients as compared with that of the control or de novo AML bone marrow. 22 Therefore not only the proliferation of MDS clone cells but also the abnormal function of stromal cells of the bone marrow may be involved in mechanisms causing cytopenias in MDS through disturbed regulation of the expression and production of growth factors and cytokines.…”

Macrophages accumulate in the early phase of tendon–bone healing

“…It is likely that macrophages play an important role in tendon healing since these cells function to scavenge cellular debris following injury and they can release growth factors that induce extracellular matrix synthesis [13] and stimulate fibroblasts or other stem cells to proliferate [5]. Previous studies of wound healing in other tissues clearly indicate that macrophage accumulation is an early event in the healing process [21]. For example, Wright et al recently reported that macrophage-lineage cells accumulate in a healing medial collateral ligament injury in a mouse model [28].…”