Neutrophils are the first inflammatory cells to arrive to the site of injury where their primary role is phagocytosis of pathogens and debris(29
). While their absolute necessity for the healing of sterile surgical wounds is debated(30
), there is clear evidence that neutrophil dysfunction leads to increased infectious complications(32
). Previous studies have demonstrated phenotypic alterations of circulating neutrophils in response to catecholamine stimulation(17
), however the effect of these stimuli on recruited wound neutrophils is unknown(39
). The present study is the first to demonstrate norepinephrine-mediated alterations in the process of phagocytosis by wound neutrophils.
We have demonstrated that exogenous pharmacologic-dose norepinephrine decreased the phagocytic efficiency of neutrophils isolated from wounds 120 hours post-injury. Our results also indicate that norepinephrine-mediated suppression of wound neutrophil phagocytosis involves both the α- and β-adrenergic receptors and intracellular signaling via protein kinase A, the primary target of cAMP (). Previous reports of catecholamine modulation of neutrophil phagocytosis have focused on exercise as a trigger of the stress response(40
). Studies of peripheral blood neutrophils isolated from human subjects before and after exercise, as well as examination of exogenous catecholamine treatment on these cells, demonstrate that norepinephrine stimulates phagocytosis, and that this stimulation involves both α- and β-adrenoreceptor activation(22
). Indeed, there is a large body of literature demonstrating catecholamine-mediated stimulation of adrenoreceptors, elevation of intracellular cAMP and signaling via its associated protein kinases in neutrophils(13
Putative model of norepinephrine-mediated alterations in 120-hour wound neutrophil phagocytosis
We observed that neutrophils isolated from 24-hour wounds, unlike those from 120-hour wounds, did not demonstrate norepinephrine-responsiveness of their phagocytic processes. This is in contrast to circulating 24-hour and 120-hour neutrophils from restraint-stressed animals, which display quantitatively similar alterations in phagocytosis at both time points(45
). A number of possibilities for this temporal difference in the wound neutrophil response to norepinephrine exist. When comparing wounds versus time, a smaller percentage of cells from 120-hour wounds undertook phagocytosis than those at 24-hours. It is possible that this group of cells represents a subset of total wound neutrophils that exhibit norepinephrine-responsiveness(46
), but does not comprise a large enough percentage of the 24-hour wound population for this difference to be detected. However, a previous, selective examination of neutrophil subsets from whole blood failed to demonstrate differences in catecholamine response(28
). Alternatively, there is evidence that the pattern of gene expression of wound neutrophils differs from that of circulating cells, and these changes may evolve over time in the wound(26
). Additionally, a temporal difference in catecholamine response may reflect a change in the neutrophils’ function in the wound over time. There is evidence that macrophage-produced tumor necrosis factor primes neutrophils for enhanced responsiveness to IL-8 through negative modulation of cAMP(47
). In wound neutrophils from later time points, the altered catecholamine-responsiveness of phagocytosis may reflect these changes in cAMP signaling. The molecular basis for this difference between circulating and tissue neutrophils is unclear, but may be related to inflammatory priming(48
). Circulating neutrophils demonstrate increased surface expression of L-selectin, a marker of cellular maturity, in response to catecholamines. However, there is shedding of this marker upon infiltration to the wound and during ongoing inflammation(49
The model that we employed in order to investigate the catecholamine response of wound neutrophils is likely to influence the results that we have observed. In this system, wound inflammatory cells are isolated in aggregate, and no further attempts are made to separate individual cell types. As would be expected, neutrophils were the predominant cell type in the wounds at 24 hours (45±7.5%), with macrophages predominating at 120 hours (41±2%). While this is likely a better model of in vivo
effects of catecholamines on these cell types, it does make comparison of our results to those of groups employing purified cell populations less straightforward. Injury and sepsis result in the production of marked amounts of both pro- and anti-inflammatory cytokines by neutrophils and macrophages and multiple studies have demonstrated alterations in the production of pro-inflammatory cytokines in response to catecholamines(50
). While it is difficult to develop a consistent framework of catecholamine/cytokine/phagocytosis interactions because of the heterogeneity of cell types and doses used in various studies, recent work indicates that catecholamine effects on target cells are dependent upon the cellular level of cyclic AMP and alteration of gene transcription via cAMP-reponse element binding (CREB) protein(51
A handful of studies have examined alterations in neutrophil phagocytosis following sympathectomy(55
). While these studies differ in the technique of sympathectomy employed, in all cases the phagocytic function of peripheral blood neutrophils was assessed and compared between sympathectomized subjects and un-treated controls. Each group of investigators observed a decrease in phagocytosis by neutrophils in the sympathectomy group. These results further augment the observation that exogenous catecholamines enhance peripheral blood neutrophil phagocytic function(23
). Additionally, these studies provide a nice counterpoint for our observation that, following sympathectomy, wound neutrophils demonstrated increased phagocytosis (unpublished observation), which is consistent with the present finding that exogenous norepinephrine suppresses wound neutrophil phagocytic function.
Following phagocytosis, neutrophils kill pathogens with antimicrobial proteins and production of reactive oxygen species such as superoxide, hydrogen peroxide and hypochlorous acid(29
). Because these same fundamental biochemical processes that govern microbial killing by neutrophils are capable of damaging host cells and tissue(20
), neutrophils are implicated as the mediators of tissue injury in a variety of inflammatory disorders(58
). In addition to modulating the process of phagocytosis, catecholamines may represent a target for the manipulation of these potentially detrimental neutrophil functions(24
), and further investigation of these processes is warranted.
Our results reinforce a role for norepinephrine in the modulation of the phagocytic processes of neutrophils. Importantly, our observations in concert with those of other groups, point towards important functional differences between circulating and wound neutrophils as well as temporal differences between early and late wound neutrophils. Examination of associated neutrophil functions, such as production of reactive oxygen species, as well as changes in the neutrophil transcriptome as a potential final common pathway for alterations in phagocytosis and catecholamine-responsiveness may yield targets for future work in the therapeutic manipulation of innate immunity to influence wound healing.