We present here a detailed characterization of macrophage extracellular trap formation. Bovine macrophages produce extracellular traps in a dose- and time-dependent manner in response to M. haemolytica
and its LKT ( and ). MET formation peaked at a 3-fold increase, which is similar to results for NET formation published by other investigators in response to various pathogens (35
). We used several methods to confirm that LKT, and not contaminating LPS, was responsible for MET formation. These included the use of (i) cytochalasin D, to inhibit LKT-mediated internalization (2
); (ii) antibodies to LKT and its CD18 receptor; (iii) heat-inactivated LKT, heat-inactivated M. haemolytica
cells, or heat-inactivated ΔlktC
cells (100°C for 1 h); (iv) a truncated ΔLKT; and (v) purified M. haemolytica
LPS alone (). Taken together, these data indicate that LKT causes MET formation via binding to its CD18 receptor and that M. haemolytica
LPS (25 nM to 2.5 μM) by itself does not cause MET formation by bovine macrophages (data not shown). Similar results were reported using bovine neutrophils (4
). The lack of LPS-mediated extracellular trap formation is similar to the results of Clark et al. (18
), who reported that LPS alone did not stimulate NET production by human neutrophils, unless platelets were also present. However, we cannot completely exclude a role for LPS as a cofactor in the MET response to M. haemolytica
cells or to LKT.
Greater concentrations of RTX toxins can disrupt the cytoplasmic membrane and cause lysis of host cells (47
). To examine what was responsible for the release of DNA, we quantified LDH release, which is a marker for necrosis (21
). LDH release remained low at LKT concentrations (0.25 to 2 U) that trigger MET formation, indicating that necrosis was not responsible for extracellular DNA. However, LDH release did increase when macrophages were incubated with a higher dose (5 U) of LKT (D and E). These data are similar to those presented by Pilsczek et al. (35
). These investigators observed little LDH release during NET formation, which they interpret to be evidence that NET formation does not lead to neutrophil lysis. Rather, these investigators observed that NETs are found in budding vesicles that burst upon leaving the neutrophil (35
). We infer from these results that low concentrations of LKT (<5 U) cause extracellular DNA release and MET formation by a process that is largely independent of necrosis.
Interestingly, extracellular traps were produced by macrophages in response to either native fully active LKT or nonacylated pro-LKT (C). This observation is similar to that reported for bovine neutrophils (4
). These data suggest that binding of pro-LKT to CD18 is sufficient to trigger extracellular trap formation by bovine leukocytes in the absence of an overt cytotoxic effect, as detected by standard cytotoxicity assays (4
). MET formation was inhibited by preincubation of LKT or pro-LKT with a neutralizing anti-LKT antibody (C). MET formation in response to LKT or pro-LKT was reduced when macrophages were preincubated with an anti-CD18 antibody or cytochalasin D, the latter of which we have previously shown inhibits intracellular trafficking of LKT (2
) (C). Overall, these data confirm that the interaction between LKT or pro-LKT and the CD18 receptor and the subsequent reorganization of intracellular actin are required for MET formation in response to LKT or pro-LKT.
We used monocyte-derived macrophages (cultured in vitro
for 7 days) for most of our experiments. Likewise, bovine alveolar macrophages also formed METs in a dose- and time-dependent manner in response to M. haemolytica
(). These data are consistent with earlier reports that alveolar macrophages exhibit streaming nuclei during M. haemolytica
infection of cattle (1
). It is interesting that maximal MET formation by alveolar macrophages occurred somewhat later (30 min) than that observed with monocyte-derived macrophages (). However, it should be noted that a limited number of alveolar macrophage samples were tested (n
= 3). In contrast to the findings described above, freshly adherent monocytes did not form extracellular traps when incubated with LKT (data not shown). These findings suggest that the maturation state of bovine mononuclear phagocytes influences their release of nuclear DNA in response to LKT.
Investigators have reported that NET formation occurs within 10 min to 4 h (35
), whereas eosinophils produce extracellular traps within seconds (45
). We found that MET formation occurred faster than NET formation, with significant accumulation of extracellular DNA occurring within 2 min after the addition of stimuli to macrophages (D and E) (4
). This appears to be similar to findings of a recent study that demonstrated NET formation at as early as 5 min in response to S. aureus
). The mechanisms by which extracellular traps are formed by various types of leukocytes have not been firmly established. However, the temporal differences in DNA release suggest that the intracellular signaling that regulates MET and NET formation may differ.
We used confocal and scanning electron microscopy to analyze the structure of METs. Confocal analysis confirmed that extracellular DNA fibrils were released in response to LKT or M. haemolytica
cells, and these fibrils were disrupted by the addition of DNase I (A). SEM confirmed the presence of a large network (>10 μm2
) of DNA fibrils that, upon higher magnification, clearly contained M. haemolytica
cells trapped within them (). The origin of DNA in extracellular traps is generally thought to be nuclear, although it is reported that eosinophils release mitochondrial DNA (48
). We addressed this issue by examining the colocalization of TOPRO, which stains DNA, with the signal from a fluorescently conjugated antibody that stains nuclear histones. The rationale for this approach is based on histones being associated with nuclear but not mitochondrial DNA (14
). Colocalization of the signal for TOPRO (red) and the antihistone antibodies (green) suggests that the DNA in METs that bovine macrophages produced in response to M. haemolytica
and its LKT is of nuclear origin (C). Incubation with DNase I confirmed that extracellular staining of TOPRO was specific for DNA.
NET formation by human neutrophils is dependent on NADPH oxidase activity (10
). Neutrophils from chronic granulomatous disease patients, whose leukocytes do not produce NADPH oxidase, do not form NETs in response to PMA or S. aureus
). Here, we provide evidence that bovine MET formation is also dependent on NADPH oxidase activity. We tested two chemicals (PMA and glucose oxidase) reported to stimulate NET formation (21
) and observed MET formation in response to each (). PMA stimulates production of NADPH oxidase through activation of protein kinase C, while glucose oxidase causes NET production without activation of NADPH oxidase (21
). Conversely, preincubation of macrophages with DPI, an NADPH oxidase inhibitor, reduced MET formation in response to M. haemolytica
cells (B). Interestingly, bovine macrophages required higher concentrations of PMA and GO to stimulate MET formation and higher concentrations of DPI to inhibit M. haemolytica
-induced MET formation than those previously reported for human NET formation () (21
We found that METs trap and kill M. haemolytica cells in a time-dependent manner. Coincubation of M. haemolytica-stimulated macrophages with DNase I reduced the numbers of M. haemolytica cells trapped and killed (A and B), indicating that cleavage of extracellular DNA released bacterial cells snared within the DNA fibrils and hence reduced MET-mediated killing. The mechanism by which extracellular traps kill bacteria is not clear. Because trap formation relies on glucose oxides or NADPH oxidase activity, local release of reactive oxygen intermediates may contribute to bacterial death. We also speculate that antimicrobial proteins (histones and others) enmeshed in the DNA fibrils might contribute to bacterial killing. Because LKT is secreted by M. haemolytica cells, we hypothesized that macrophages might be exposed to LKT prior to contact with M. haemolytica cells during natural infection. We explored this possibility and found that preincubation with LKT increased the numbers of M. haemolytica cells that were trapped and killed by METs (C and D). We speculate that release of extracellular traps by bovine macrophages and neutrophils in response to LKT is an effort by the host to localize and impede invasion by the pathogen. However, one might envision that M. haemolytica cells which are trapped, but not killed, by METs could release LKT and LPS to stimulate local inflammation. Whether MET formation enhances the trapping and killing of bacterial cells in vivo or provides a niche in which surviving M. haemolytica cells release LKT and LPS to augment inflammation in the lung remains to be answered.
Other researchers found that only a minority (~10 to 40%) of neutrophils form NETs when incubated with a stimulus (21
). Using confocal microscopy to quantify cells forming traps, we observed similar percentages of macrophages undergoing MET formation in response to LKT, M. haemolytica
cells, or PMA (B). It should be noted that a single cluster or strand of DNA was scored as one MET, although it is possible that more than one macrophage could have contributed to that DNA staining.
Earlier histopathology studies revealed streaming leukocytes resembling extracellular traps within the alveoli of cattle with M. haemolytica
). Extensive extracellular DNA was found in M. haemolytica
-infected lung tissue (4
), although the origin of the DNA was not determined. We have observed colocalization of extracellular DNA and the macrophage marker MIP-1α in histological sections from M. haemolytica
-infected lung tissue (data not shown). These observations do not provide direct evidence for MET formation in vivo
but are consistent with prior reports that some streaming leukocytes might be leukocytes (including macrophages) that have formed extracellular traps (12
The relevance of this study is broadened by evidence that extracellular trap formation is not restricted to bovine macrophages. Using the mouse macrophage cell line RAW 264.7 and the human monocyte cell line THP-1 (), we observed MET formation in response to a related RTX toxin, the HLY from a uropathogenic strain of E. coli
). LKT did not induce MET formation in these cell lines (data not shown). Unlike LKT, whose activity is specific for ruminant leukocytes, HLY is active against leukocytes from many species (33
). Cytochalasin D did not reduce MET formation by RAW 264.7 or THP-1 cells in response to HLY (). This result is consistent with our previous finding that HLY intoxication of bovine lymphoblastoid (BL-3) cells is unaffected by cytochalasin D and, hence, independent of host cell actin rearrangement (unpublished observations). Overall, these data suggest that MET formation in response to RTX toxins is not restricted to bovine macrophages and is representative of a broader response exhibited by mouse and human macrophages.
While preparing the manuscript, Chow et al. reported MET formation by RAW 264.7 cells and thioglycolate-elicited murine peritoneal macrophage cells in response to S. aureus
cells. A portion of the S. aureus
cells was killed in this process (17
). Treatment of RAW 264.7 cells or murine peritoneal macrophages with statins enhanced MET-mediated S. aureus
). Formation of METs by human monocytes and macrophages in response to gold nanoparticles was also recently reported (5
). Whether extracellular traps are beneficial or detrimental to host defense is unclear at this time. Although the potential beneficial effect of trapping and killing extracellular bacterial cells is obvious, the presence of NETs has recently been linked to several human disease states, including systemic lupus erythematosus (27
), peritonitis (42
), vasculitis (30
), septic arthritis (31
), and pre-eclampsia (25
). What role macrophage extracellular traps play in host defense and the pathophysiology of infectious diseases will be the subject of future investigations.