Phagocytic capacity, microbial killing and TNF release

Although it has been reported that severe trauma contributes to an increase in infectious complications through an impairment of host defence mechanisms ¹⁶³, the cause of such impairment remains unknown ¹⁶⁴. The alveolar macrophage, mobile representative of the mononuclear phagocyte system, is believed to be the central modulator, both as regulator and effector of inflammation/ anti-inflammation within the alveolar space ⁴⁸.

In the present study, blast pressure wave exposure resulted in an immediate decrease in the alveolar macrophage (AM) phagocytosis rate of Escherichia coli (E. coli) in vitro with recovery within 96 hours after blast. Although not statistically significant, the phagocytic capacity for Staphylococcus aureus (S. aureus) particles seemed to be reduced initially after thoracic trauma. Disorders of the phagocytic process in alveolar macrophages have been reported after thermal injury ^226,227. These studies showed a primary decrease occurring immediately after the burn. The discrepancy between the different phagocytosis rates of both bacterial stimuli may be explained by experimental problems rather than by strains differences.

Whereas the E. coli particles were suspended homogenously, the S. aureus particles formed aggregates despite of sonicating them and this may affect particle counts and therefore the read-out of the experiment. The use of different lots of fluorescence labelled particles may also contribute to the controversial results. Nevertheless, as macrophages are the primary resident phagocyte of the resting human lung, it is intuitive that any functional impairment leads to infection by micro-organisms. Thus, despite recovery within 4 days after trauma, these data suggest an initially impaired phagocytosis capacity due to thoracic trauma, that may result in a higher susceptibility to infections. It is possible that trauma-induced factors or mediators may either bind to the macrophage surface or suppress macrophage activity, thereby down-regulating phagocytosis. It is also conceivable that impairment of the pulmonary surfactant during lung injury contributes to a decreased phagocytosis capacity. Indeed, surfactant protein A ⁷¹ and D ⁷² are able to precoat bacteria and particles in order to facilitate macrophage phagocytosis and bactericidal activities. The trauma-related, deteriorated tidal volume and pulmonary compliance we assessed ex vivo in our lung perfusion model support such a trauma-induced loss in surfactant.

The production of reactive oxygen species (ROS) (“respiratory burst”) upon receptor-mediated phagocytosis was investigated to determine the microbicidal activity of the AM in vitro after in vivo thorax trauma. Thoracic trauma yielded a marked decrease in the production of superoxide within the first 24 hours after trauma, both with or without the addition of E. coli or S. aureus, with recovery only after 96 hours. The respiratory burst results from the assembly

Discussion

either by a decrease in its production or by an increase in its elimination, be it through interfering with one or more steps in the signal transduction pathway at either the cell membrane level or subsequent intracellular stage such as dismutase, protein kinase (PK)-C activation or through scavenging by other radicals, such as nitric oxide. With regard to this, it has been shown that LPS-related oxidative burst and TNF-release by human monocytes can be modulated by IL-9 through an upregulation of TGF-β which in turn inactivates the extracellular signal-regulated kinases (ERK) 1/ 2 ⁸⁴. TGF-β is involved in bronchial wound repair ²²⁸.

The difference between trauma and control in both the resting and the activated state, could therefore arise from both a decreased activity of one or more oxidases and an increased ability of the cells to eliminate superoxide, due to cellular damage and released NO (discussed in 5.3.2.3).

Since overnight culturing of the isolated control AM reduced the spontaneous superoxide production to about 30% of its initial value, the increased release of superoxide in non-traumatized controls may be attributed to the activation of NADPH oxidase during the macrophage isolation procedure. In contrast, the superoxide anion levels produced upon E.

coli stimulation were not affected by overnight culturing in these cells and thus may be related to the bacterial stimulus (data not shown). In order to preserve the in vivo conditions after trauma in terms of both macrophage environment and time-dependent AM behaviour, we abstained form overnight culturing before stimulation.

Similarly, trauma-related initial decrease of superoxide generation was independent of the presence of lipopolysaccharide (LPS) or lipoteichoic acid (LTA). In contrast to the stimulation with whole dead bacteria, upon LPS/ LTA stimulation the superoxide levels of non-traumatized controls were reached already at 24 hours, and were increased 96 hours after the blast. Since the levels of superoxide produced by the non-stimulated controls were different between the experiments, the higher respiratory burst capacity observed upon LPS/ LTA stimulation as compared to E. coli/ S. aureus stimulation, may be due to an increased basal stimulation, rather than to their particular specificity. In contrast to this stimulus-independent effect, the earlier recovery together with the increased oxidative burst after 96 hours, suggests higher susceptibility of the AM towards LPS and LTA as compared to E. coli and S. aureus stimulation in this setting. However, the most likely explanation for this would be that the amount of surface-bound endotoxin on E. coli and LTA on S. aureus particles is not comparable with the amount of LPS and LTA used in these experiments. This explanation is further supported by the fact that both whole bacteria and endotoxin, act on the same receptors on the macrophage surface. Recent studies have identified multiple receptors for endotoxin which has accelerated the study of signalling in macrophages (reviewed by Monick and Hunninghake, ²²⁹, Wright, ⁶⁶). Two classes of receptors, the CD18 antigens (leukocyte

Discussion

integrins) and the scavenger receptor (acetyl low-density lipoprotein), recognize particulate LPS directly. Both these receptors may be involved in catabolism of LPS, because neither receptor initiates strong secretory responses e.g. TNF synthesis to LPS. A third receptor, CD14, recognizes LPS complexed with the serum lipoprotein binding protein (LBP) ⁶². CD14 appears to participate in both the ingestion of, and the responses to LPS, because a blockade of CD 14 with monoclonal antibodies (mAbs) strongly inhibits both uptake of bacteria and secretion of TNF by human mononuclear cells ⁶². A recent study suggested that the recognition sites of CD14 for LPS and LTA are distinct, with a partial overlap ²³⁰.

Furthermore, leukocytes express three different receptors for complement protein C3, and 10 distinct receptors for the Fc region of IgG ⁶⁰. With regard to possible regulatory mechanisms of phagocytosis and oxidative burst in the present study, Pricop and Salmon ⁷⁷ reported that FcγR activation induces the NADPH oxidase and thus increases ROS formation. These authors further demonstrated that ROS generated in an inflammatory milieu act in an autocrine and paracrine manner in order to rapidly amplify the effector potential of FcγR on quiescent phagocytes by altering signal transduction.

We further investigated macrophage TNF release after experimental thorax trauma, alone and in response to either endotoxin/ LTA or whole dead bacteria. The TNF release of AM from traumatized rat lungs was increased 96 hours after blast as compared to non-traumatized controls, indicating that the AM are primed to release TNF following thoracic trauma. This result is in line with increasing TNF levels measured in BAL fluids of traumatized rat lungs in vivo over time (discussed in 5.3.2.2). However, when compared to the early TNF release measured in BAL fluids from 3 hours after trauma on, the TNF release by isolated AM in vitro is delayed. Additional stimulation of the AM with either endotoxin/ LTA or bacteria induced a marked increase in TNF release 24 hours after blast exposure, whereas initially after blast the response of the AM towards both stimuli was comparable or even decreased (E. coli stimulation) with that from AM of non-traumatized controls. We conclude that thoracic trauma primes AM to release TNF which can be augmented by an additional bacterial stimulus. With regard to TNF release, AM from sham controls as well as from traumatized rats were more susceptible to LPS than to LTA (up to 8-fold and 7-fold, respectively at the highest used concentration) and more to E. coli than to S. aureus stimulation (up to 4.7-fold and 6.5-fold, respectively). This is in close correlation to reports from Morath and colleagues ²³¹, who described LTA as a weak inducer of cytokine release in human blood compared to LPS.

Many experimental models of trauma have demonstrated a change in macrophage function as

Discussion

after injury. The causes of abnormal macrophage function appear to be multifactorial but are not well understood. Injury causes a stress response which in turn has been recognized as a source of immunosuppression. Thus, physical or even psychological stresses trigger the hypothalamic-pituitary-adrenal (HPA) axis, resulting in a marked rise in serum levels of many hormones e.g. glucocorticoids ¹³⁰. Receptors for glucocorticoids are present on macrophages

233 and have been shown to downregulate macrophage functions ²³². Furthermore, not only stress but also pro-inflammatory cytokines such as IL-1, IL-6 and TNF cause via the release of corticotropin the release of glucocorticoids (reviewed in ¹³⁰). Thus, glucocorticoid-mediated immunosuppression following injury may represent dysfunction of normal, inhibitory feedback pathways. With regard to this, Cech and colleagues ²³² reported increased plasma corticosterone levels one day after femure fracture in mice. In this study femur fracture resulted also in a reduction of 1. Macrophage production of superoxide anion, 2. C. albicans killing, 3. macrophage synthesis of IL-6 and TNF and 4. an increase in PGE₂ synthesis. These authors further demonstrated that pre-treatment with the glucocorticoid receptor antagonist, mifepristone (RU 486) significantly prevented or reduced the observed suppression of several macrophage functions, including oxidative burst capacity and C. albicans killing but did not alter the increased PGE₂ synthesis. However, the initially suppressed macrophage function was supposed to be at least in part due to the trauma-related PGE₂ production ¹⁵⁵. The results from our study demonstrated a recovery from an initially suppressed E. coli phagocytosis and oxidative burst within 24 hours and 96 hours, respectively. Such a rebound phenomenon has also been described for peritoneal macrophages after laparotomy ¹⁵⁶. We further identified a hyperactive TNF response of AM to endotoxin/ LTA and whole bacteria stimulation that is seen 24 hours after blast exposure. This is in line with McCarter and colleagues ¹⁵⁵ who found increased TNF, IL-6 and H2O2 secretion of splenic macrophages in response to endotoxin or PMA stimulation 7 days after trauma. These authors further demonstrated that inflammatory hypersecretion does not necessary translate into improved immunologic protection, as demonstrated by the functional impairment of macrophage antigen presentation. It is likely that the impairment of phagocytic function and microbicidal activity after thoracic trauma observed in this setting could contribute to a depressed antigen presentation. However, preliminary findings showed that Keyhole Limped Hemocyanin (KLH)-sensitised rats developed a cutaneous anergy during about four days after severe trauma, which underlines that the intensity of the investigated trauma has systemic repercussions and seems to include aspects of immunosupression.

The search of the signalling endotoxin receptor led to the discovery of Toll receptors (reviewed by Monick and Hunninghake, ²²⁹): TLR 4 is the LPS-specific receptor, whereas TLR 2 has been linked to LTA from Gram-positive bacteria. Both TLR 2 and TLR 4 have been found on alveolar macrophages, providing the entry point for a complex cascade of LTA/ LPS signalling.

Discussion

Downstream of the TLR 4 complex, a number of signalling pathways are activated. These include the MAP kinases, phosphatidylinositol (PI) 3-kinase pathway and sphingolipid metabolites that regulate LPS-induced gene expression. Protein kinase (PK) C and PI3-kinase have been shown to be not only involved in the phagocytosis of E.coli particles, but also in the signalling pathway of LPS-induced TNF release. In contrast, anti-inflammatory phosphodiesterase inhibitors prevented LPS-induced TNF release but had no effect on the beneficial phagocytosis of E. coli by macrophages ²³⁴.

5.4.3

Involvement of trauma-related alveolar haemorrhage in impaired