Murine polymorphonuclear neutrophils produce interferon- in response to pulmonary infection with Nocardia asteroides

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1 Murine polymorphonuclear neutrophils produce interferon- in response to pulmonary infection with Nocardia asteroides Terri N. Ellis and Blaine L. Beaman Department of Medical Microbiology and Immunology, University of California School of Medicine, University of California, Davis Abstract: Nocardia asteroides causes an acute, necrotizing pneumonia characterized by extensive infiltration of polymorphonuclear neutrophils (PMNs) into the lungs. Although PMNs have historically been classified as end-point cells, recent investigations have indicated that PMNs have the ability to secrete cytokines such as interleukin (IL)-4 and IL-12. This study investigated the ability of PMNs to produce cytokines in a murine model of N. asteroides pulmonary infection. Flow cytometric analysis demonstrated the production of interferon- (IFN- ), but not IL-4, by PMNs in response to this infection. IFN- production correlated with peak infiltration of PMNs into the lungs. Cell sorting and enzyme-linked immunosorbent assay were used to confirm cytokine production by cells with nuclear morphology characteristic of PMNs. This is the first report of IFN- production by neutrophils in response to an infection in vivo. These results suggest that PMNs play an important role in directing the host toward a T helper cell type 1 phenotypic response in the lungs. J. Leukoc. Biol. 72: ; Key Words: cytokines granulocytes flow cytometry inflammation INTRODUCTION Polymorphonuclear neutrophils (PMNs) have historically been described as terminally differentiated cells of the immune system. This description implies that PMNs function as phagocytes to degrade foreign entities and release nonspecific mediators of inflammation, such as lysozyme and leukotrienes, from preformed granules [1]. Secretion of specific cell signaling molecules, such as cytokines, was thought to be the role of cells involved in an antigen-specific response, including macrophages, B cells, or T cells. Recently, this view has been challenged, as PMNs have been demonstrated to secrete cytokines. In vitro studies established the ability of PMNs to produce a wide range of cytokines such as interleukin (IL)-4, IL-12, and tumor necrosis factor (TNF- ) [2 4]. These observations are being confirmed in vivo, as reports of cytokine production by PMNs in the context of infections are increasing [5, 6]. Nocardia asteroides is a facultative, intracellular pathogen that causes an acute pulmonary infection characterized by necrotizing pneumonia and extensive infiltration of PMNs into the lungs. This initial accumulation of PMNs is followed by an influx of mononuclear cells that effect clearance of the bacteria [7]. Research has indicated that neutrophils, although a critical component of the response to N. asteroides, are not capable of effecting bacterial clearance without other cell types. Studies have demonstrated that PMNs alone are not able to kill N. asteroides, but rather inhibit the growth of the organism [8]. In this situation, PMNs may be an important source of signaling molecules to call in a more effective response composed of mononuclear cells. In this study, we investigated the ability of PMNs to produce cytokines during the course of pulmonary infection with N. asteroides strain GUH-2. The data demonstrate that infiltrating PMNs secrete interferon- (IFN- ) during the course of infection. Although secretion of IFN- by PMNs has been documented in vitro with artificial stimulation, this study appears to be the first report of this phenomenon in vivo in response to an infectious agent. MATERIALS AND METHODS Mice C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Female mice aged 5 8 weeks, housed in pathogen-free rooms, were used for all experiments. The University of California, Davis, Animal Care and Use Committee approved all experimental protocols. Bacterial infection N. asteroides strain GUH-2 was grown to mid-log phase in 50 ml brain heart infusion broth (BHI). A suspension of single cells at an optical density of 0.7 (580 nm) was prepared by differential centrifugation and resuspended in BHI. Mice were anesthetized with an intraperitoneal injection of 50 mg/kg Nembutal (Abbot Labs, Chicago, IL), and 50 L prepared bacterial dose was given by intranasal aspiration. Mice mock-treated with 50 l BHI broth were used as controls. Three hours postinfection, the lungs of four infected mice were harvested, homogenized, and plated on BHI agar to determine the effective dose received in the lung. The effective dose in the lungs for all experiments was colony-forming units. Histology At each time point, control and infected mice were sacrificed, and the lungs were perfused with 10% neutral-buffered formalin in phosphate-buffered sa- Correspondence: Blaine L. Beaman, Department of Medical Microbiology and Immunology, University of California School of Medicine, University of California, Davis, CA blbeaman@ucdavis.edu Received November 17, 2001; revised March 20, 2002; accepted March 25, Journal of Leukocyte Biology Volume 72, August

2 line via a tracheal cannula. Fixed lungs were paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Observations were made using light microscopy at 400 magnification. Isolation of lung cells and whole blood At each time point, control and infected mice were sacrificed, and the lungs were excised. The lungs were then manually disrupted with a 3-cc syringe in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and sieved through a 100- cell strainer to remove debris. The number of viable cells in suspension was quantified using trypan blue exclusion, and cell concentrations were standardized for use in the following assays. Whole blood was obtained from control (BHI-treated) animals after cervical dislocation. Intracellular flow cytometry The following protocol was adapted from the protocol for Activation and Intracellular Staining of Whole Blood for Flow Cytometry, found in the Cytokine/Chemokine Application Manual (Pharmingen, San Diego, CA). Lung cells or whole blood were incubated for 4hat37 C in RPMI 1640 with 10% FCS and brefeldin A, a Golgi complex inhibitor, to allow for accumulation of intracellular cytokines. Following incubation, cells were treated with Pharm- Lyse reagent (Pharmingen) to lyse red blood cells and were stained for surface markers. The antibodies used for surface staining were flourescein isothiocyanate (FITC)-conjugated anti-mouse Ly-6G (Gr-1), FITC or phycoerythrin (PE)- labeled anti-mouse I-A b major histocompatibility complex (MHC) class II and natural killer (NK) 1.1, and peridinin chlorophyll protein-conjugated rat anti-mouse CD3ε. Samples were also stained with appropriately conjugated, irrelevant isotype antibodies as a negative control (all antibodies from Caltag, Burlingame, CA, or Pharmingen). Cells were then permeabilized using the Caltag Fix and Perm kit and were stained with PE-conjugated rat anti-mouse IFN- or IL-4 antibodies. Cells were gated based on forward versus side scatter, and 5000 events were collected. Data were analyzed on a Becton Dickinson FACScan using CellQuest software (Becton Dickinson, San Jose, CA). Fluorescence-activated cell sorting (FACS) and histology The isolated suspension of lung cells was treated with PharmLyse reagent and labeled with FITC-conjugated anti-mouse Ly-6G, which labels PMNs. Labeled cells were sorted using a MoFlo FACS (Cytomation, Fort Collins, CO). Viability of sorted cells was quantified by trypan blue exclusion. Cells were prepared by cytocentrifugation and differentially stained using the Hema 3 Wright stain kit (Biochemical Sciences Inc., Swedesboro, NJ) for observation under a light microscope at 400 magnification. Enzyme-linked immunosorbent assay (ELISA) For ELISA, cells from lung suspensions ( cells/ml) were incubated overnight at 37 C in RPMI 1640 with 10% FCS. Supernatants of cell cultures were then used to assay for IFN- secretion using the OptEIA cytokine ELISA kit (Pharmingen), according to the manufacturer s instructions. Statistical analysis Statistical significance was determined using Student s t-test. Significance was defined as P RESULTS Kinetics of PMN infiltration during infection Pulmonary infection with N. asteroides GUH-2 causes an acute pneumonia characterized by PMN infiltration. Figure 1 illus- Fig. 1. Histology of murine lungs (H&E stain) following intranasal infection with N. asteroides GUH-2. (A) 24 h postinfection, (B) 3 days postinfection, (C) 5 days postinfection, and (D) 7 days postinfection. 374 Journal of Leukocyte Biology Volume 72, August

3 trates that PMNs are the predominant cell type in the infiltrate through 3 days postinfection (Fig. 1, A and B). By 5 7 days, this infiltrate changed to a predominately mononuclear response. To quantify the cell types involved in this infiltration, a suspension of single cells was prepared from the lungs of infected mice at four time points during the course of infection. Flow cytometric analysis of these suspensions was performed to determine the relative numbers of CD3 T cells and polymorphonuclear neutrophils in the lung. Results of a representative experiment, shown in Figure 2A, demonstrate the rapid infil- Fig. 2. Flow cytometric quantitation of PMN influx during pulmonary infection with N. asteroides GUH-2. Relative numbers of PMNs and CD3 T cells were determined at 1, 3, 5, and 7 days postinfection. Gates were set by forward versus side scatter, and 5000 events were collected. (A) Representative dot plots of each time point for infected and BHI control mice. Percentages of positively stained cells within the gate (5000 events) are shown in relevant quadrants. (B) Average levels of PMNs in the lung over the course of infection as determined by flow cytometry. Each time point consists of two to three independent experiments with n 4 for each experiment. *, Statistical significance from the control value as determined by Student s t-test (P 0.05). Error bars represent standard error from the mean. Ellis and Beaman PMNs produce IFN- in response to Nocardia 375

4 tration of large numbers of PMNs into the lungs (upper left quadrant) as compared with uninfected control mice. Figure 2B shows the average infiltration as determined over three separate experiments. High levels of PMNs were present in the lung by 24 h postinfection, and this level was maintained at 3 days postinfection. By 5 days postinfection, PMNs had begun to dissipate and by 7 days postinfection, had returned to control levels. At this time, the bacteria had been cleared from the lung (data not shown). The levels of CD3 T cells did not change significantly during the course of infection (data not shown). Production of IFN- during infection The production of cytokines during infection with N. asteroides GUH-2 was assayed by flow cytometry. Lung cells from infected mice were collected at 1, 3, 5, and 7 days postinfection. Fig. 3. Representative dot plots of IFN- production during pulmonary infection with N. asteroides GUH-2 at 1, 3, 5, and 7 days postinfection. (A) Flow cytometric detection of intracellular IFN- levels in CD3 T cells of infected (left) and BHI-treated (right) mice. (B) Flow cytometric detection of intracellular IFN- levels in polymorphonuclear neutrophils of infected (left) and BHI-treated (right) mice. Percentages of positively stained cells within the gate (5000 events) are shown in each quadrant. 376 Journal of Leukocyte Biology Volume 72, August

5 These suspensions of single cells were then cultured for 4 h in the presence of brefeldin A, a Golgi transport inhibitor, to allow intracellular cytokines to accumulate to detectable levels. These cells were then labeled with FITC-conjugated surface antibodies to CD3 or Ly-6G (a PMN-specific glycoprotein), permeabilized, and stained with PE-conjugated antibody to IFN- or IL-4. Figure 3 shows representative dot plots of IFN- production. Averaged levels are shown in Figure 4. As seen in Figure 3A, CD3 T cells, the traditional source of IFN-, did not produce significant levels of this cytokine. However, significant levels of IFN- can be seen in Figure 3A in the CD3-negative population, and Figure 3B identifies the IFN- -producing population as PMNs. Figure 4 shows the average levels of production of IFN- (A) and IL-4 (B). By flow cytometry, significantly high numbers of IFN- -producing cells were present at 24 h and 3 days postinfection. IL-4-producing cells were not observed at any point during the course of infection (Fig. 4B). The LY-6G antibody used here to positively identify PMNs does cross react to a small extent with macrophages. To eliminate the possibility that the cross-reacting population is responsible for cytokine production, cells were stained with antibody to MHC class II molecules. Because macrophages, but not PMNs, are antigen-presenting cells, this permits the two populations to be easily distinguished. As seen in Figure 5A, the majority of infiltrating cells did not express MHC II. Figure 5B demonstrates that IFN- was produced solely by those cells that are negative for MHC II. NK cells, another cell type that commonly produces IFN-, were also assayed for cytokine production. NK 1.1 and LY-6G were not observed to be coexpressed on cells (Fig. 5C) nor were NK cells observed to produce IFN- at any time during infection (Fig. 5D). Of all cell types tested, which included CD3 cells (T cells), NK 1.1 cells, and MHC II cells (monocytes/ macrophages), only those cells with a PMN profile (LY-6G ; all other listed markers, negative) exhibited IFN- production. Additionally, peripheral blood PMNs from uninfected mice were not observed to stain with the IFN- antibody (Fig. 6), confirming the specificity of the antibody. Flow cytometry data indicated IFN- production by PMNs, but did not indicate active secretion of the cytokine. Therefore, secretion of IFN- was confirmed by ELISA. As shown in Figure 7, significantly high levels of IFN- were detected at 1, 3, and 5 days postinfection. This pattern of cytokine production matches the pattern of PMN infiltration seen in Figures 1 and 2. Because PMNs were observed to be the only cell type producing IFN- during this infection, it can be concluded that PMNs produced the IFN- detected by the ELISA. IFN- secretion by highly purified PMNs To demonstrate directly that PMNs were actively secreting IFN- during the course of infection, PMNs were purified from the lungs of mice 3 days postinfection. Cell suspensions of total lung cells were stained with FITC-labeled Ly-6G, and the PMNs were purified using a MoFlo FACS. The resulting suspension was determined to be at least 95% pure by flow cytometry (data not shown). Aliquots of this purified sample were then taken for microscopic analysis and culture. Figure 8 shows the morphology of these cell suspensions before (A) and after (B, C) cell sorting. Sorted cells exhibited the characteristic nuclear morphology of PMNs (C). These purified cells were cultured overnight, and the supernatants were assayed for IFN- secretion by ELISA. As seen in Figure 8D, the purified PMNs from infected mice produced statistically significant levels of IFN-. The small increase in IFN- seen in the sorted, uninfected cells was not statistically significant from the unsorted control. DISCUSSION Current research into the function of PMNs has revealed that these cells are able to actively synthesize and secrete proteins while acting as phagocytes. The data presented here demon- Fig. 4. Average levels of IFN- (A) and IL-4 (B) produced by PMNs and CD3 T cells as determined by flow cytometry. Bars indicate average levels of cytokine production by CD3 T cells and PMNs from control and infected mice. Each time point consists of two to three independent experiments with n 4 for each experiment. *, Statistical significance from the control value as determined by Student s t-test (P 0.05). Bars represent standard error from the mean. Ellis and Beaman PMNs produce IFN- in response to Nocardia 377

6 Fig. 5. Representative dot plots of MHC II or NK1.1 expression during pulmonary infection with N. asteroides GUH-2 at 1, 3, 5, and 7 days postinfection. (A) Relative numbers of cells positive for LY-6G (PMN marker) and MHC II. (B) Flow cytometric detection of IFN- levels in MHC II-positive cells. (C) Relative numbers of cells positive for LY-6G (PMN marker) and NK 1.1 (NK cells). (D) Flow cytometric detection of IFN- levels in NK 1.1-positive cells. Percentages of positively stained cells within the gate (5000 events) are shown in each quadrant. strate that PMNs actively secrete IFN- during the course of pulmonary infection with N. asteroides GUH-2. To our knowledge, this is the first report of IFN- being produced by PMNs in response to a pathogen. Yeaman et al. [9] recently demonstrated the ability of human PMNs to produce IFN- when cultured and artificially stimulated ex vivo. Our studies confirm this previous report and demonstrate this activity in the context of an in vivo infection. This study adds IFN- to the list of (primarily proinflammatory) cytokines and chemokines that can be produced by PMNs. The data presented here indicate that PMNs respond to N. asteroides by producing IFN-. The peak of secreted IFN- was observed at 3 days postinfection (Figs. 4 and 7), when large numbers of PMNs were present (Fig. 2). When comparing 378 Journal of Leukocyte Biology Volume 72, August

7 Fig. 6. Flow cytometric detection of IFN- levels in peripheral blood PMNs from uninfected mice. Percentages of positively stained cells within the gate (5000 events) are shown in each quadrant. Figures 4 and 7, the IFN- levels at 5 days postinfection appear to be quite different. However, this may be explained by methods of detection. Flow cytometry detects the number of cells producing IFN-, and ELISA detects extracellular, secreted cytokines. Therefore, although a small population of PMNs actively secreting high levels of IFN- would only be measured as a small number of IFN- cells by flow cytometry, the large amount of secreted product would be detected by ELISA. Extensive control assays were performed to eliminate the possibility of other cell types contributing to IFN- production (Figs. 5 and 6). In all assays performed, PMNs were the only cell type observed producing IFN- during the course of this infection. The sorted cells assayed in Figure 8 were sorted to a purity of at least 95%. This does leave open the possibility that the other 5% of cells present in the culture are responsible for the IFN- production observed. However, given that PMNs were the only cell type observed producing IFN-, this is an unlikely scenario. Although all samples were cultured at comparable concentrations, the drop in IFN- production by the infected, sorted group as compared with the infected, unsorted could be because of cellular trauma during the cell sorting process that may lead to some cell death during culture. Regardless, the infected, sorted samples produced significantly higher levels of IFN- than the uninfected control. These data confirm that IFN- was actively secreted by PMNs during pulmonary infection with N. asteroides GUH-2. The levels of IFN- detailed here are low when compared with reported levels produced by mononuclear cells. This is a common phenomenon of cytokine production by PMNs. In a survey of cytokine production in standardized numbers of PMNs and mononuclear cells, Cassatella [10] found that for six out of nine cytokines assayed, significantly lower levels were produced by PMNs than mononuclear cells. Large numbers of PMNs, often the predominate infiltrating cell type, may individually produce low levels of a cytokine such as IFN-. owever, the large numbers of infiltrating PMNs may result in an additive effect that is comparable with that seen by fewer mononuclear cells producing higher levels of cytokines [10]. It should be noted that the levels of IFN- presented here (180 pg/ml; Fig. 7) are seven times higher than those presented by Yeaman et al. [9]. The values presented by Yeaman et al. [9] were modestly above the detection limit (30 pg/ml) of the ELISA kit used in our studies. This observation may indicate that N. asteroides is a potent stimulator of cytokine production in PMNs. Experiments to determine the direct effect of N. asteroides on PMN cytokine production are ongoing. Research into the immune response to N. asteroides GUH-2 may partly explain one reason for cytokine production by PMNs. Previous studies have shown that neutrophils alone are not capable of killing Nocardia. In vitro incubation of N. asteroides with purified neutrophils did not significantly decrease the number of viable bacteria, although the bacteria had been phagocytized and exposed to the oxidative burst [11, 12]. Although PMNs were shown to inhibit the growth and metabolic processes of phagocytized N. asteroides, they were not effective at killing this pathogen [8, 13]. Because the neutrophils are unable to fully clear the bacterial infection, other cells must be recruited. Our data suggest that PMNs may play an important role in this recruitment and activation process. Neutrophils may be reacting directly to the viability of the phagocytosed N. asteroides via cytokine production to modulate the immune response. During N. asteroides infection, the traditional, primary functions of phagocytosis and oxidative burst are ineffective. This may drive neutrophils to actively secrete signaling molecules to call in and modulate a more effective response. Five days postinfection appeared to be a transition time, as mononuclear cells began to infiltrate into the lung (Fig. 1). This observation correlates with research done on the role of T cells during this infection. As reported by Tam et al. [14], T cell numbers peak at 5 days postinfection with N. asteroides GUH-2. This transition was reflected in the data presented Fig. 7. Levels of secreted IFN- in the lung as determined by ELISA. Aliquots of total lung cells were incubated overnight in RPMI 1640 with 10% FCS. Supernatants were assayed for IFN- production by ELISA using the Pharmingen OptEIA kit. Effective limit of detection was 30 pg/ml. Bars represent standard error from the mean. Ellis and Beaman PMNs produce IFN- in response to Nocardia 379

8 Fig. 8. FACS of PMNs in lung 3 days postinfection with N. asteroides GUH-2. Aliquots of cells before (A) and after sorting (B) were prepared by cytocentrifugation, stained with the Hema 3 Wright s stain kit, and observed under a light microscope at 400. (C) Enlargement of representative cells post sorting. (D) IFN- secretion by total lung cells (crude Unsorted) and PMNs purified from mouse lung supernatants 3 days postinfection with N. asteroides GUH-2. Aliquots of total lung cells and PMNs purified by FACS were incubated overnight in RPMI 1640 with 10% FCS. Supernatants were assayed for IFN- production by ELISA using the Pharmingen OptEIA kit. *, Statistical significance from the control value as determined by Student s t-test (P 0.05). Error bars represent standard error from the mean. here by the fact that at 5 days postinfection, PMN numbers were declining, but those still resident produced some IFN-. It may be at this time that other cytokines are produced to fine tune the mononuclear cell response in which T cells have been shown to play an important role. [14, 15]. This increase in T cells may also explain why an increase in total CD3 T cells was not observed in response to this T helper cell type 1 cytokine. We have observed that rather than a total CD3 T cells increase during 5 7 days postinfection, the proportion of these cells shifts from T cells to primarily T cells (unpublished observations). We are investigating what other signals may be involved in this response and if there is a connection between PMN-derived cytokines and the T cell response. The results presented here were obtained without secondary stimulation during culture. However, these PMNs were stimulated in vivo by a natural pathogen. This is in contrast with the results of Yeaman et al. [9], who used PMNs derived from healthy donors and artificially stimulated in culture. By artificial stimulation, Yeaman et al. [9] found that PMNs produced IFN- in response to lipopolysaccharides (LPS), IL-12, or TNF-. The results of Yeaman et al. [9] would appear to contradict that of Keel et al. [16], who found that PMNs stimulated with LPS in vitro do not produce IFN-. The data presented here are the first of IFN- being produced by PMNs in vivo, in response to a pathogen. It should be noted that N. asteroides is a gram-positive nocardioform actinomycete that lacks LPS [7]. However, the complex cell wall of Nocardia, like that of Mycobacterium tuberculosis, contains many potent, nonspecific stimulators of the immune system, including mycolic acids and other glycolipids. We are investigating the molecular signals from the bacteria that may be stimulating PMNs to produce IFN-. The number of microbial infections that has been demonstrated to involve PMN cytokine production is growing. Examples include Pseudomonas aeruginosa, eliciting IL-8 from PMNs [17], Candida albicans infection resulting in IL-10 and IL-12 production by PMNs [18, 19], and Mycobacterium avium infection, resulting in TNF-, IL-12, and IL-1 production [6]. Additionally, studies of lung fibrosis using a bleomycin mouse model have indicated the importance of IFN- from a non-t cell source during inflammation [20]. These studies suggest that the importance of PMN cytokine production has neither been fully appreciated nor explored. Romani et al. [18] demonstrated the necessity of PMNderived cytokines in the context of C. albicans infection. During Candida infection, PMN-derived IL-12 was shown to be critical for survival of the host through development of an effective response. Neutrophil-depleted mice did not survive infection unless given exogenous IL-12, which enabled the animals to survive and clear the organism in a manner similar to wild-type animals. The discovery that PMNs may secrete such powerful and critically required, activating cytokines, such as IL-12 and IFN-, and not just chemoattractants attests 380 Journal of Leukocyte Biology Volume 72, August

9 to the importance of PMNs in the response to and the modulation of the immune response. Many of the signaling molecules documented to be produced by PMNs are chemokines, such as IL-8 and macrophageinflammatory protein-1 [21], which serve primarily to attract other cells to a site of infection. However, our data demonstrate that PMNs also have the ability to produce the powerful, activating cytokine IFN-. Immune modulator molecules, such as IFN-, were long considered to be secreted predominantly by T cells. The ability of PMNs to secrete these cytokines and call in other cell types as the host response progresses indicates that PMNs can play an active role in modulating and communicating with the immune system. The data presented here indicate that the role of PMNs goes beyond simple recruitment of cells to that of activation and modulation of function. 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