These studies demonstrate the surprising and unexpected finding that neutrophils are producing IFN-γ early in the inflammatory response during pneumonia induced by some bacteria, but not others. Although the possibility that another cell type also contributes to IFN-γ production by this 24-hour time point cannot be completely excluded, the only CD45+ leukocyte producing IFN-γ expresses the neutrophil-specific surface marker, Ly6G (19). S. pneumoniae and S. aureus induced the production of IFN-γ, whereas P. aeruginosa and E. coli did not. Neither LPS nor LTA, two lipid substances within the wall of gram-negative and gram-positive organisms, respectively, induced IFN-γ production by neutrophils, suggesting that other components of bacteria were regulating IFN-γ production either directly or through host defense molecules. IFN-γ was produced only by neutrophils that had emigrated into the tissue at 24 hours and was not present at 6 hours postinfection. Neither circulating neutrophils nor neutrophils within the pulmonary microvasculature that had not yet migrated expressed IFN-γ. Whether the process of migration is critical for IFN-γ production or whether the time needed to migrate is required for the production of this cytokine remains a question.
IFN-γ production was documented by three different approaches. Flow cytometry was used to document intracellular IFN-γ and the cell type that was producing it. To confirm that the anti–IFN-γ antibody was specifically identifying intracellular IFN-γ, the antigen recognized by the antibody was determined by mass spectroscopy and confirmed to be murine IFN-γ. The presence of IFN-γ in the tissue was documented by sensitive ELISAs. Last, IFN-γ mRNA was produced by neutrophils. These data clearly document that neutrophils were producing IFN-γ and that this cytokine was expressed early in the inflammatory response to these two gram-positive organisms.
Our studies are not the first to demonstrate that IFN-γ is produced by neutrophils. In 1998, Yeaman and colleagues demonstrated that neutrophils in the stroma of the uterus stain for IFN-γ at all stages of the cycle, as do circulating neutrophils from the blood treated ex vivo
with IL-12 (3
). Kirby and colleagues demonstrated that splenic neutrophils and macrophages produced IFN-γ in response to Salmonella
). During early graft loss of pancreatic islet transplants, NK T cells stimulate production of IFN-γ by neutrophils (10
). Early in renal ischemia–reperfusion injury, NK T cells also induced expression of IFN-γ in neutrophils within the kidney (9
). Furthermore, Ellis and Beaman demonstrated that neutrophils within the lungs produced IFN-γ in response to Nocardia asteroides
). Most recently, Nance and colleagues demonstrated that neutrophils generate IFN-γ mRNA and protein in response to intrapulmonary Saccharopolyspora rectivirgula
, an infection that mimics hypersensitivity pneumonitis (39
). Our studies are the first to identify neutrophils as a source of IFN-γ in acute bacterial pneumonias, to demonstrate that neutrophils transcribe mRNA, and to address both the function of IFN-γ and the mechanisms through which it is regulated.
These studies also examined the pathways required to produce this IFN-γ. The nonreceptor Src tyrosine kinases Fgr/Hck/Lyn, the small GTPase Rac2, and the NADPH oxidase component gp91phox
were also each required for the production of IFN-γ. Other studies have demonstrated that outside/in intracellular signaling initiated by ligation of CD18 can result in the activation of these Src kinases (40
). In turn, these Src kinases activate Rac2 (25
), which is required for activity of the assembled NADPH oxidase complex, resulting in the production of oxidants (26
). Whether this CD18:Fgr/Hck/Lyn:Rac2:NADPH oxidase pathway is the signaling pathway critical in the production of IFN-γ, or whether each of these molecules is critical in other pathways resulting in IFN-γ production, remains to be determined. However, our data clearly show that Rac2, Fgr/Hck/Lyn, and NADPH oxidase are each required for IFN-γ production by neutrophils. Taken together, these data suggest that production of oxidants and the result oxidative stress may regulate production of IFN-γ mRNA.
Curiously, all four organisms, as well as LPS and LTA, result in the production of IFN-γ mRNA, but only S. pneumoniae and S. aureus resulted in the expression of protein from the message. These data suggest that the translation of IFN-γ mRNA to protein is the critical step in the mechanism underlying this differential expression. Our studies examined the hypothesis that differential expression of microRNAs targeted to the noncoding region of IFN-γ may be the mechanism through which translation is differentially regulated, by quantifying the expression of 17 microRNAs identified through database searches as having sequences that potentially recognize the noncoding region of the IFN-γ mRNA and are produced by neutrophils. Our results show that eight microRNAs are induced in E. coli but not S. pneumoniae pneumonias ( and ), and the expression of these microRNAs correlates with the lack of translation of IFN-γ mRNA. None of these 17 microRNAs was differentially induced only in S. pneumoniae pneumonia, in which both IFN-γ mRNA and protein are produced. Although these data do not prove that these microRNAs are the mechanism for the differential expression of IFN-γ protein from mRNA induced by S. pneumoniae and S. aureus compared with E. coli, P. aeruginosa, LPS, and LTA, they do provide evidence that this may in fact be the mechanism. This inhibition of translation may be due to the cumulative effect of the 56–228% increases in these differentially expressed microRNAs. Furthermore, five of the eight differentially expressed microRNAs target sites within region 375–412 of the IFN-γ gene (), which may also enhance the effect of each. Moreover, the ability of E. coli to induce the production of these microRNAs targeted to IFN-γ may represent a novel strategy by which this pathogen can evade host defenses, which could also be shared by other pathogens toward IFN-γ or other host genes.
How the genes for these microRNAs are regulated is an important question. The gene miR-106b is regulated by the E2F1 transcription factor (42
). The expression of miR-15a and miR-26a is modulated by c-Myc (43
). However, to our knowledge, these are the only studies examining the regulation of these differentially regulated miR genes, and determining whether a common regulator might be increased in E. coli
but not S. pneumoniae
pneumonia is not yet possible until more information about the transcription factors modulating these genes is available.
Most importantly, our studies demonstrate that IFN-γ production modulates host defense against pneumonia. Bacterial clearance of S. pneumoniae
(2.8–5.0 × 106
cfu/mouse) from the lungs is eightfold less in IFN-γ–deficient compared with wild-type mice. This poor clearance is accompanied by an increase in neutrophils, which is most likely the result of increased numbers of organisms. In contrast, during E. coli
pneumonia, in which IFN-γ is not produced, there is no difference in bacterial clearance between these two genotypes. Studies to understand how IFN-γ is altering bacterial clearance demonstrate that the production of NETs was highly dependent on the production of IFN-γ. NETs are an important bactericidal mechanism in neutrophils, as important or more important than phagocytosis in sepsis and likely in later stages of the neutrophil life span (27
). Studies have demonstrated that neutrophils from neonates have no defect in IL-8 production but do have a defect in NET formation compared with neutrophils from adults, and this defect correlated with a deficit in extracellular bacterial killing (45
). In addition, the production of NETs was much greater in response to S. aureus
than E. coli in vitro
), which, when considered in light of our observation that S. aureus
but not E. coli
induces production of IFN-γ, further suggests that IFN-γ is required for robust generation of NETs. Thus, our studies suggest that IFN-γ is regulating the formation of NETs, a highly bactericidal effector mechanism by neutrophils, and that this NET formation is responsible for the poor clearance of S. pneumoniae
in IFN-γ–deficient animals. Other functions for IFN-γ also seem likely, for example, in regulating the balance between Th1 and Th2 responses and the production of granulomas (7
In summary, these studies demonstrate that neutrophils produce IFN-γ in response to S. pneumoniae
and S. aureus
pneumonias in mice and that this IFN-γ production is important in host defense and the clearance of organisms from the lungs. These studies add to a growing list of critically important functions of neutrophils not only in releasing preformed granular contents and in generating reactive oxygen species but also in synthesizing regulatory molecules that modulate the inflammatory and innate immune responses (48
). IFN-γ production is highly regulated, and these studies begin to identify the signaling molecules that are important in the production of IFN-γ by these cells. The clinical relevance of the production of IFN-γ by neutrophils at this early time in the evolution of bacterial pneumonia is demonstrated by the defect in host defense that results when IFN-γ cannot be produced. These studies also suggest that host defense may be enhanced by the administration of exogenous IFN-γ in susceptible patient populations. In fact, IFN-γ is already used in patients with chronic granulomatous disease, in which NADPH oxidase is genetically dysfunctional. Thus, the induction of IFN-γ and its function have important therapeutic implications.