Phagocytosis is a phylogenetically conserved process by which cells ingest microbial pathogens and debris(24
). While a number of different cell types can undertake phagocytosis, macrophages are professional phagocytes, a specialized subset of immune cells that respond to activation and differentiation signals by modulating their phagocytic efficiency(25
). Norepinephrine, the primary neurotransmitter of the peripheral sympathetic nervous system, is released in large quantities from peripheral nerves into the circulation following injury and has previously been shown to be involved in wound healing(26
). While a number of studies report catecholamine-mediated alterations in the phenotype of splenic and peritoneal macrophages(20
), the present study is the first to delineate a role for norepinephrine in modulation of wound macrophage phagocytic function.
We have demonstrated that exogenous norepinephrine, in both physiologic and pharmacologic doses, decreased wound macrophage phagocytic efficiency. Interestingly, there was no alteration in the number of macrophages that engaged in phagocytosis, a quantity that is often reported in the literature to be altered by norepinephrine treatment(31
). Our results also indicate that this norepinephrine-mediated effect acts through both the α- and β-adrenergic receptor pathways in a dose-defined fashion. We observed that lower doses of norepinephrine acted primarily through the β-adrenergic receptors while higher doses had a predominantly α-adrenergic mechanism of transmission (). This result is in contrast to previous reports of norepinephrine modulation of cytokine function(15
) but is consistent with the previous observation that norepinephrine competes for β-adrenoreceptor binding with relatively low potency(14
) and evidence that high doses of catecholamines may lead to internalization of the β-adrenoreceptor(32
Putative model of norepinephrine-mediated alterations in wound macrophage phagocytosis
A distinction between resident and recruited macrophages is recognized in the literature(33
). We examined the tissue-bed specificity of norepinephrine-mediated modulations in macrophage phagocytosis by comparing wound and splenic macrophages isolated from the same animals at the same time point. We observed that, at baseline, splenic macrophages demonstrate lower phagocytic efficiency than wound macrophages. Additionally, norepinephrine did not alter phagocytosis by splenic macrophages from wounded animals. The underlying reasons for tissue-bed variation in macrophage responsiveness are unclear and multiple possibilities exist. Alterations in surface receptor expression during the process of monocyte to macrophage differentiation may result in varied phagocytic ability of macrophage populations in different anatomic sites(35
). Evidence for this theory includes data that appearance of αV
integrin on the surface of cells is temporally connected to their transition from monocytes to macrophages in vitro
, and this parallels the appearance of phagocytic capacity(36
). Another possibility is that wound macrophages represent a subset of monocytic cells that are able to respond to norepinephrine, and that splenic macrophages with this ability have been mobilized from the spleen to the site of injury(37
). The activation state of macrophages is influenced by changes in the wound microenvironment, and may result in alterations of phagocytic capacity as well(38
). Additionally, alterations in relative density of α- and β-adrenoreceptors on the macrophage surface could contribute to the observed differences(39
), but this concept has not been adequately explored in the literature.
Further evidence for the concept of tissue bed specificity of the macrophage response comes from studies of splenic, peritoneal and alveolar macrophages. Examination of catecholamine influence on phagocytosis by splenic macrophages reveals findings both complementary to and conflicting with ours(21
). These investigators demonstrated a suppression of both percent phagocytosis and phagocytic efficiency in splenic macrophages isolated from animals that had experienced restraint stress. Additionally, in vitro
norepinephrine treatment of splenic macrophages from unrestrained animals demonstrated a dose-response relationship, with enhancement of phagocytosis seen with low doses (10−11
M) and suppression seen with higher doses (10−5
M). These responses were abrogated by β-adrenergic blockade but were independent of α-adrenergic blockade. In contrast, previous studies of the role of catecholamines in modulating phagocytosis by peritoneal macrophages demonstrated a β-adrenoreceptor dependent enhancement of phagocytosis from doses of norepinephrine in the 10−5
Studies of alveolar macrophages have demonstrated predominantly inhibitory effects of cAMP on phagocytosis, generation of reactive oxygen intermediates and the production of inflammatory cytokines(40
). More recently, investigation of the modulation of alveolar macrophage function has revealed differential roles for protein kinase A (PKA) and exchange protein directly activated by cAMP-1 (Epac-1), the downstream targets of cAMP(19
). Specifically, suppression of phagocytosis appears to be mediated by Epac-1, but not PKA. The reverse is true of alveolar macrophage production of pro-inflammatory mediators, such as leukotriene B4
and TNF-α, which appears to be suppressed by PKA activation. In our study, suppression of phagocytic efficiency was observed with direct elevation of intracellular cAMP and a complete reversal of norepinephrine-mediated suppression was seen with PKA blockade. This would appear to point away from a role for Epac-1 in norepinephrine-mediated changes in phagocytosis by wound macrophages.
Taken as a whole, our results and those of other groups indicate a definite role for norepinephrine in the modulation of phagocytosis and phagocytic efficiency by macrophages. While heterogeneity in specific tissue-bed responses is present, the fundamental mechanisms involved include stimulation of either α- or β-adrenoreceptors followed by intracellular signaling via cAMP and its dependent protein kinases. Further evaluation of downstream changes in the gene transcription as a potential final common pathway for alterations in phagocytosis is warranted. These results reinforce the concept of norepinephrine and its intracellular signaling mediators as potential therapeutic targets in the manipulation of innate immunity.