Molecular and Metabolic Evidence for the Restricted Expression of Inducible Nitric Oxide Synthase in Healing Wounds

Abstract

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in woundsonly during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10 , most macrophages in the wound were negative for iNOS. These cells , however , acquired iNOS antigen and activity in culture. Wound fluids , but not normal rat serum , suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound. (Am J Pathol 1999, 154:1097-1104)Previous work described a temporally restricted pattern of activity for two distinct enzymes of L-arginine metabolism, arginase and the inducible form of nitric oxide synthase (iNOS), in healing wounds. 1,2 Arguing from the accumulation of specific L-arginine catabolites in extracellular fluid obtained from experimental wounds and in cultures of whole-wound explants, it was proposed from this laboratory that the expression of iNOS in acute wounds was restricted to the period of polymorphonuclear leukocyte (PMN) infiltration, which encompasses the initial 24 to 72 hours after injury. Results also indicated that macrophage-derived arginase was the preponderant, and probably the only, high-flux enzyme of arginine catabolism in the wounds thereafter. Additional support to the latter conclusion and an enzymatic basis for the virtual disappearance of L-arginine from the extracellular space of late wounds were given by the accumulation of arginase in extracellular fluids obtained from wounds of increasing maturity.1,2 Additional studies identified other wound-associated microenvironmental factors, such as hypoxia, as decidedly preferential in inducing L-arginine metabolism in macrophages through arginase rather than iNOS. 3,4The relevance of the product of iNOS, NO, and its putative derivatives to inflammatory processes in general and more specifically to wound healing has recently been highlighted. 5 It has been proposed, in this regard, that the sustained expression of NOS in healing wounds is critical to the accumulation of collagen and the acquisition of mechanical strength in wounds.5 These results were, interestingly, obtained in part usi...

Bacterial Colonization and the Expression of Inducible Nitric Oxide Synthase in Murine Wounds

“…9 Current results differ from those reported previously from this laboratory in rat incisional wounds. 1 In those wounds, iNOS protein and the metabolic products of the NOS reaction (eg, citrulline) were detectable in wound cells and wound fluids during the initial, inflammatory phase of the repair process. The difference in the expression of iNOS in wound cells of rats and mice is probably explained by the relative promiscuity of expression of iNOS in rat versus mouse inflammatory cells.…”

“…Macrophages contributed the bulk of the iNOS protein to the wound, with the remainder found in polymorphonuclear leukocytes. 1 Based on such findings, it was proposed that NO generated by iNOS could participate in the regulation of the inflammatory phase of wound healing.…”

Dietary Glycine Inhibits Angiogenesis During Wound Healing and Tumor Growth

“…Macrophages are known to be the major source of iNOS production during wound healing and their expression of iNOS is highest at the granulation tissue phase. 35 Macrophages have been implicated in all aspects of wound healing and angiogenesis. 36 During the granulation tissue phase, they are the predominant cell type and are responsible for production of cytokines and growth factors essential to the healing cascade.…”

Macrophage-Induced Neutrophil Apoptosis

“…Recent work from this laboratory demonstrated that inducible NO synthase is expressed exclusively by M in day 1 wound cell preparations (27). Because NO is known to induce apoptosis in target cells (28), its potential role in M-dependent PMN apoptosis was investigated.…”