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The Journal of Immunology, 2007, 179: 1942-1949.
Copyright © 2007 by The American Association of Immunologists, Inc.
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IFN-{gamma}-Mediated Survival Enables Human Neutrophils to Produce MCP-1/CCL2 in Response to Activation by TLR Ligands1

Teizo Yoshimura2 and Munehisa Takahashi

Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLRs are key elements of the pathogen recognition mechanism used by the host immune system. Neutrophils express almost all TLRs, and activation of TLRs, such as TLR2 and TLR4, has been shown to induce the production of proinflammatory cytokines and chemokines, potentially linking innate and adaptive immunity. In the present study, we investigated whether activation of TLRs induces neutrophil production of MCP-1/CCL2, a key mediator involved in the development of adaptive immunity. Activation of neutrophils with LPS, lipoteichoic acid, or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Ser-[S]-Lys did not induce significant MCP-1 production and release; however, the Th1 cytokine IFN-{gamma} dramatically up-regulated MCP-1 production in cells activated with each TLR ligand. The majority of MCP-1 was released between 24 and 48 h of culture, indicating that this is a late event. The effect of IFN-{gamma} appeared to be due to its antiapoptotic effect, but not priming effect, revealing a biological consequence of IFN-{gamma}-induced neutrophil survival. Although IFN-{gamma} failed to protect neutrophils from cell death at a higher dose of LPS, the p38 MAPK inhibitor SB203580 dramatically increased MCP-1 release and neutrophil survival at this LPS concentration. Thus, p38 MAPK plays a previously uncharacterized role in neutrophil function. Taken together, our results indicate that human neutrophils produce MCP-1 in a Th1 microenvironment and this neutrophil-derived MCP-1 potentially amplifies the development of Th1 adaptive responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutrophils are the most abundant leukocytes, comprising about two-thirds of peripheral blood leukocytes in humans. Upon tissue injury, they rapidly infiltrate injury sites and play an important role in innate immune responses. In addition, there is accumulating evidence indicating a role for neutrophils in the development of adaptive immune responses. Neutrophil depletion in mice resulted in impaired Th1-mediated adaptive immune responses, such as delayed-type hypersensitivity reaction (DTH) to Ags, such as SRBC (1). More recently, neutrophil-depleted mice exhibited lower levels of the Th1 cytokines IFN-{gamma} and IL-12 and elevated levels of the Th2 cytokines IL-4 and IL-10 in the lungs of a Legionella pneumophila pneumonia model, suggesting a role of neutrophils in Th1 polarization to microbial Ags (2).

Neutrophils can contribute to the development of adaptive immune responses through two mechanisms. One is by functioning as APCs and the other is by producing proinflammatory cytokines and chemokines. In fact, neutrophils have the capacity to express MHC class II molecules and have been reported to present Ags to T cells (3, 4, 5). Additionally, highly purified, lactoferrin-positive, immediate precursors of end-stage neutrophils were shown to be capable of reversing their functional maturation program and to be driven to acquire the characteristics of dendritic cells (DC)3 (6). In our previous study, however, cytokine-activated, fully mature neutrophils expressed cell surface molecules, such as MHC class II, CD40, CD83, and CCR6, but they did not activate allogeneic MLR and did not express CCR7, indicating that although cytokine-activated neutrophils acquire some features characteristic of DC, mature neutrophils do not parallel the maturation program of DC (7, 8). It is more likely that mature neutrophils contribute to the process by producing cytokines and chemokines (9, 10).

MCP-1/CCL2 is a chemokine regulating the recruitment of monocytes to sites of tissue injury and plays a critical role in the development of adaptive immune responses, especially of Th1-type responses. A previous study using a rat DTH model demonstrated that early infiltrating neutrophils were the main source of MCP-1, and neutralization of this chemokine inhibited the infiltration of monocytes and T cells and subsequent development of DTH (11). We and others have shown that human neutrophils are capable of producing MCP-1 in vitro in response to the products of mononuclear leukocytes or GM-CSF (12, 13). Therefore, neutrophil-derived MCP-1 is a candidate molecule responsible for neutrophil-mediated amplification of the adaptive immune responses.

TLRs are key initiators of innate and adaptive immune responses through production of proinflammatory cytokines and chemokines, up-regulation of costimulatory molecules, and activation of Ag presentation. Neutrophils express a wide variety of TLRs, including TLR1, 2, 4, 5, 6, 7, 8, 9, and 10 (14, 15, 16). Activation of TLR4 with LPS induces the production of several proinflammatory cytokines and chemokines, including IL-1beta, TNF-{alpha}, and several chemokines, such as IL-8 (9). In the present study, we evaluated whether human neutrophils produce MCP-1 in response to activation of TLR2 or TLR4. Activation of neutrophils with TLR2 or TLR4 ligands did not result in the production of MCP-1; however, interestingly, addition of the Th1 cytokine IFN-{gamma} dramatically up-regulated the production of MCP-1 by prolonging its survival. Our results support the role for neutrophils in the development of adaptive immune responses, particularly of the Th1 responses. We also demonstrate a novel role for p38 MAPK in neutrophil survival and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

RPMI 1640 and TRIzol reagent were obtained from Invitrogen Life Technologies. FCS was purchased from HyClone. Recombinant human TNF-{alpha} and IFN-{gamma} were obtained from PeproTech. A neutralizing mouse mAb against human TNF-{alpha} (clone 1825.121) was purchased from R&D Systems. Dextran T-500 was obtained from Pharmacia Biotech. Accu-Prep was purchased from Accurate Chemical & Scientific. [{alpha}-32P]dCTP was obtained from Amersham Biosciences. LPS was purchased from Sigma-Aldrich. LTA and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Ser-[S]-Lys (Pam3CSK4) were obtained from InvivoGen.

Preparation of neutrophils

Human neutrophils were obtained from heparinized blood from human donors (10 U of heparin/ml of blood). One volume of 5% dextran (T-500; Pharmacia) in PBS was added to three volumes of blood in 50-ml polypropylene tubes. After 40 min of incubation at room temperature, the leukocyte-rich plasma layer was removed and overlaid onto Lymphoprep in 50-ml tubes, and the tubes were centrifuged at 800 x g for 20 min at room temperature. Neutrophils were separated from erythrocytes by lysis in 0.2% NaCl, washed in complete medium three times at 4°C, and resuspended in complete medium. PBMC contained in the neutrophil preparations obtained by this method were <0.5% (<5 PBMC/1000 cells) by morphologic examination. Cell viability was higher than 98% by trypan blue staining. In some experiments, PBMC fractions were also collected, washed once with PBS at room temperature and twice with RPMI 1640 containing 10% FCS (complete medium) at 4°C, and resuspended in complete medium.

ELISA

Neutrophils were cultured at a density of 5 x 106/ml in 1 ml of complete medium in the presence or absence of different stimuli in 12-well plates (Costar). After incubation for various periods of time at 37°C, cell-free culture supernatants were obtained. The concentrations of MCP-1 were measured in the Lymphokine Testing Laboratory, Clinical Services Program, SAIC-Frederick, National Cancer Institute-Frederick (Frederick, MD) with an ELISA kit specific for human MCP-1 (R&D Systems). The sensitivity threshold of the assay was 15.6 pg/ml.

Northern blotting

Northern blot analysis was performed as described previously (17) in 1.2% agarose gels containing formaldehyde. Membranes were hybridized at 42°C overnight in 50% formamide, 5x SSC, 5x Denhardt’s solution, 50 µg/ml sheared-denatured salmon sperm DNA, 1% SDS, and lx 106 dpm/ml 32P-labeled human MCP-1 cDNA probe (18). Filters were washed twice with 2x SSC/0.5% SDS at room temperature for 15 min and 0.1x SSC/0.5% SDS at 60°C for 30 min before autoradiographic exposure.

Assay for apoptosis

One milliliter of neutrophil suspension at the cell density of 5 x 106 cells/ml was plated into each well of 12-well tissue culture plates in the absence or presence of cytokines or LPS. After a 24-h incubation at 37°C, the cells were collected and the exposure of phosphatidylserine was evaluated by the FACScan flow cytometer (BD Pharmingen) using the Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen).

Statistical analysis

Statistical analysis was performed by one-way ANOVA, followed by the Bonferroni multiple comparison test, using the GraphPad Prism version 4 (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Microbial Ags and IFN-{gamma} synergistically induce MCP-1 release from human neutrophils

To examine whether microbial Ags are capable of inducing MCP-1 production in neutrophils, we cultured human neutrophils in vitro for 24 or 48 h in the presence of LPS and quantified the concentrations of MCP-1 in the culture supernatants. As shown in Fig. 1A, there was no detectable level of MCP-1 in the culture supernatants at either 24 or 48 h.


Figure 1
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  		FIGURE 1. LPS and IFN-{gamma} synergistically induce MCP-1 expression and release in human neutrophils. A, Neutrophils were incubated in the presence or absence of LPS (1 ng/ml) and/or IFN-{gamma} (100 U/ml) for 24 or 48 h and MCP-1 concentrations in cell-free culture supernatants were measured by ELISA. Data are presented as mean ± SD of three independent experiments with three donors. A value of p < 0.05 was considered to be statistically significant. *, p < 0.001, n = 3. B, Neutrophils were incubated for 48 h in the presence of different doses of LPS in combination with 100 U/ml IFN-{gamma}. Data are presented as mean ± SD of five independent experiments with different donors. Each value was compared with that induced with 0 pg/ml LPS. A value of p < 0.05 was considered to be statistically significant. *, p < 0.05; **, p < 0.01, n = 5. C, Neutrophils were incubated for 48 h in the presence of different doses of IFN-{gamma} in combination with 1 ng/ml LPS. Data are presented as mean ± SD of five independent experiments with different donors. Each value was compared with that induced with 0 U/ml IFN-{gamma}. A value of p < 0.05 was considered to be statistically significant. *, p < 0.001, n = 5.

 
IFN-{gamma} has been demonstrated to induce the production of chemokines, such as IFN-{gamma}-inducible protein 10/CXCL10, synergistically with LPS (19). Therefore, we examined whether IFN-{gamma} could play a role in the production of MCP-1. IFN-{gamma} by itself induced only a minimal level of MCP-1. Interestingly, when neutrophils were incubated with a combination of LPS and IFN-{gamma}, significant levels of MCP-1 were detected in the culture supernatants (Fig. 1A). MCP-1 concentrations dramatically increased between 24 and 48 h, indicating that the majority of MCP-1 was released during this period. The effect of LPS was dose dependent, but 10 ng/ml LPS had a tendency to induce lower levels of MCP-1 release (Fig. 1B). One nanogram of LPS per milliliter was optimal when the IFN-{gamma} concentration was fixed to 100 U/ml. IFN-{gamma}, in combination with 1 ng/ml LPS, also dose-dependently increased MCP-1 release, as shown in Fig. 1C. From these results, 1 ng/ml LPS and 100 U/ml IFN-{gamma} were chosen for the rest of this study. In contrast to IFN-{gamma}, IL-4 showed no effect on LPS-induced MCP-1 release.

To examine whether the increased MCP-1 release was regulated at the mRNA expression level, we performed Northern blotting. Fifteen million neutrophils were incubated in the presence or absence of LPS and/or IFN-{gamma} for 4 or 16 h, total RNA was extracted, and the expression of MCP-1 mRNA was evaluated. As shown in Fig. 2A, freshly isolated neutrophils did not express MCP-1 mRNA. Incubation of neutrophils with LPS, IFN-{gamma}, or LPS plus IFN-{gamma} did not result in a detectable level of MCP-1 mRNA expression at 4 h. At 16 h, a very low level of MCP-1 mRNA was detected in cells incubated with 10 ng/ml LPS or 100 U/ml IFN-{gamma}. Interestingly, LPS and IFN-{gamma} synergistically induced a high level of MCP-1 mRNA expression at this time point. One nanogram of LPS per milliliter plus 100 U/ml IFN-{gamma} induced the highest level of MCP-1 mRNA. The levels of MCP-1 mRNA expression correlated well with those of MCP-1 concentrations detected in the culture supernatants (Fig. 1B). It should be noted that the amounts of total RNA recovered from cells incubated in medium alone or in the presence of LPS for 24 h were much less than those recovered from cells incubated in the presence of IFN-{gamma}.


Figure 2
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  		FIGURE 2. Expression of MCP-1 mRNA is up-regulated in neutrophils incubated with LPS plus IFN-{gamma}. A, Neutrophils (1.5 x 107 cells in 3 ml) were incubated for 4 or 16 h in the presence or absence of different doses of LPS (nanograms per milliliter) plus 100 U/ml IFN-{gamma}, and then total RNA was extracted. The RNA samples were loaded onto a single gel and the expression of MCP-1 mRNA was examined using Northern blot analysis. The image of the ethidium bromide-stained gel is also presented. Data are representative of four independent experiments with similar results. Contamination of PBMC was 0.2%. B, PBMC (1 x 107 cells in 2 ml) from the same donor were incubated for 4 or 24 h in the presence or absence of 1 ng/ml LPS and/or 100 U/ml IFN-{gamma} and then total RNA was extracted. The RNA samples were loaded onto a single gel and the expression of MCP-1 mRNA was examined by Northern blotting. The image of the ethidium bromide-stained gel is also presented. Data are representative of four independent experiments with almost identical results.

 
It is well known that activated PBMC is a major source of MCP-1. Although our neutrophil preparations were highly pure, it was still possible that MCP-1 protein and mRNA detected in this study were due to a contamination by PBMC. To obtain further evidence that MCP-1 mRNA detected in neutrophils was derived from neutrophils, we incubated PBMC in the presence of 1 ng/ml LPS with or without IFN-{gamma} and then examined the expression of MCP-1 mRNA by Northern blotting (Fig. 2B). Unlike neutrophils, PBMC rapidly responded to LPS and expressed a high level of MCP-1 mRNA at 4 h. There was no increase in MCP-1 mRNA expression when they were incubated in the presence of LPS plus IFN-{gamma}. IFN-{gamma} alone also induced a low but significant level of MCP-1 mRNA expression at 4 h. At 24 h, the level of MCP-1 mRNA induced with IFN-{gamma} increased, whereas the level of MCP-1 mRNA induced in response to LPS or LPS plus IFN-{gamma} decreased. Thus, the responsiveness of PBMC to LPS or LPS plus IFN-{gamma} was completely different from that of neutrophils. The kinetics of MCP-1 mRNA expression in these two cell types was also completely different. These results strongly support our conclusion that MCP-1 mRNA detected in our study was derived from neutrophils but not from contaminated PBMC. Our attempt to detect MCP-1 protein in neutrophils by ELISPOT was not successful, probably because of a low level of MCP-1 released from a single cell.

We next evaluated the capacity of two TLR2 ligands, LTA and Pam3CSK4, to induce MCP-1 release from neutrophils. As shown in Fig. 3, neither LTA nor Pam3CSK4 induced MCP-1 production; however, both TLR2 ligands, in combination with IFN-{gamma}, dose-dependently induced MCP-1 production. The levels of MCP-1 produced in response to these ligands were significantly lower than that induced with LPS plus IFN-{gamma}. These results indicated that microbial Ags, synergistically with IFN-{gamma}, were capable of inducing MCP-1 production and release in neutrophils.


Figure 3
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  		FIGURE 3. TLR2 ligands and IFN-{gamma} synergistically induce MCP-1 release in human neutrophils. Neutrophils were incubated for 48 h with different doses (nanograms per milliliter) of the TLR2 ligands LTA or Pam3CSK4 in the presence or absence of 100 U/ml IFN-{gamma}, and MCP-1 concentrations in cell-free culture supernatants were measured by ELISA. Data are presented as mean ± SD of five experiments with five donors. The values obtained with 1 ng/ml LPS or different doses of LTA or Pam3CSK4 plus 100 U/ml IFN-{gamma} were compared with the value obtained with medium alone. A value of p < 0.05 was considered to be statistically significant. *, p < 0.05; **, p < 0.01, n = 5. The values obtained with different doses of LTA or Pam3CSK4 plus 100 U/ml IFN-{gamma} were compared with the value obtained with 1 ng/ml LPS plus 100 U/ml IFN-{gamma}. A value of p < 0.05 was considered to be statistically significant. {dagger}, p < 0.001, n = 5.

 
MCP-1 release induced with LPS plus IFN-{gamma} is partly dependent on TNF-{alpha}

We previously reported that culture supernatants of PHA-activated PBMC had the capacity to induce MCP-1 production in neutrophils. We identified TNF-{alpha} as a critical factor involved in the process (13). Because activated neutrophils have been known to produce TNF-{alpha} (9, 20, 21), we hypothesized that TNF-{alpha} produced during the culture might play a role in MCP-1 production induced with LPS plus IFN-{gamma}. To test the hypothesis, we first examined whether a combination of TNF-{alpha} and IFN-{gamma} also induce MCP-1 production in neutrophils. As shown in Fig. 4A, 1 ng/ml TNF-{alpha} and 100 U/ml IFN-{gamma} synergistically induced MCP-1 release at a level comparable to that of LPS plus IFN-{gamma}. One nanogram of TNF-{alpha} per milliliter induced the highest level of MCP-1 release, whereas 10 ng/ml TNF-{alpha} induced lower levels of MCP-1 release (Fig. 4B). We next examined the role of TNF-{alpha} in MCP-1 production induced with LPS plus IFN-{gamma} (Fig. 4C). As expected, the addition of anti-TNF-{alpha}-neutralizing Ab almost completely abolished MCP-1 release induced with TNF-{alpha} plus IFN-{gamma}. In contrast, addition of this Ab reduced only 50% of MCP-1 release induced with LPS plus IFN-{gamma}, indicating that MCP-1 release induced with LPS plus IFN-{gamma} was partly, but not completely, dependent on TNF-{alpha}. The addition of normal mouse IgG1 had no effect on neutrophil MCP-1 release (data not shown).


Figure 4
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  		FIGURE 4. TNF-{alpha} is partly responsible for MCP-1 release induced with LPS plus IFN-{gamma}. A, Neutrophils were incubated in medium alone, 1 ng/ml TNF-{alpha}, 1 ng/ml TNF-{alpha} and 100 U/ml IFN-{gamma}, or 1 ng/ml LPS plus 100 U/ml IFN-{gamma} for 24 or 48 h, and MCP-1 concentrations in cell-free culture supernatants were measured by ELISA. Data are presented as mean ± SD of four independent experiments with four donors. A value of p < 0.05 was considered to be statistically significant. *, p < 0.01; **, p < 0.001, n = 4. B, Neutrophils were incubated with different doses of TNF-{alpha} plus 100 U/ml IFN-{gamma} for 48 h, and MCP-1 concentrations in cell-free culture supernatants were measured by ELISA. Data are presented as mean ± SD of three individual experiments with three donors. Each value was compared with that obtained with 0.01 ng/ml TNF-{alpha} plus 100 U/ml IFN-{gamma}. A value of p < 0.05 was considered to be statistically significant. *, p < 0.05; **, p < 0.001, n = 3. C, Neutrophils were incubated with 1 ng/ml TNF-{alpha} plus 100 U/ml IFN-{gamma} with or without different amounts of anti-TNF-{alpha}-neutralizing Ab for 48 h, and MCP-1 concentrations in cell-free culture supernatants were measured by ELISA. The percentage of MCP-1 release was calculated by comparing the concentration of MCP-1 after each treatment to that without any treatment. Data are presented as mean ± SD of three independent experiments with three donors. A value of p < 0.05 was considered to be statistically significant. *, p < 0.001, n = 3. D, Polymyxin B and/or anti-TNF-{alpha}-neutralizing Ab were added to neutrophils incubated with 1 ng/ml LPS plus 100 U/ml IFN-{gamma} at different time points. Cell-free culture supernatants were obtained after 48 h, and MCP-1 concentrations were measured by ELISA. The percentage of MCP-1 release was calculated by comparing the concentration of MCP-1 after each treatment to that without any treatment. Data are presented as mean ± SD of three independent experiments with three donors. The value obtained with a combination of anti-TNF-{alpha} Ab and polymyxin B at each time point was compared with that obtained with polymyxin B alone. A value of p < 0.05 was considered to be statistically significant. *, p < 0.01, n = 3.

 
To further examine the contribution of TNF-{alpha}, anti-TNF-{alpha}-neutralizing Ab was added 0, 3, 6, 10, or 20 h after the initiation of culture in the presence of LPS plus IFN-{gamma} (Fig. 4D). The addition of anti-TNF-{alpha}-neutralizing Ab consistently reduced MCP-1 release by ~50% up to 10 h after the initiation of culture, but ~30% when this Ab was added after 20 h of culture. Again, the addition of normal mouse IgG1 had no effect on neutrophil MCP-1 release (data not shown).

We also used polymyxin B to inhibit LPS. The addition of polymyxin B at time 0 completely inhibited MCP-1 release induced with LPS plus IFN-{gamma}; however, the effect of polymyxin B decreased gradually thereafter, and it inhibited only 30 and 20% when it was added at 10 and 20 h, respectively. Neutralization of TNF-{alpha} did not increase the inhibitory effect of polymyxin B up to 6 h; however, it provided an additional inhibitory effect when added with polymyxin B at 10 and 20 h. These results suggested that initial activation with LPS induced the production of TNF-{alpha} in neutrophils and this neutrophil-derived TNF-{alpha} provided an additional signal for the maximal MCP-1 production and release.

Initial activation with LPS, but not IFN-{gamma}, is critical for neutrophil MCP-1 release

IFN-{gamma} has been reported to prime neutrophils for a number of neutrophil functions, including oxidative burst, NO production, and phagocytosis (22). Because activation of neutrophils with LPS alone did not induce significant MCP-1 release, we hypothesized that IFN-{gamma} primes neutrophils to respond to LPS or TNF-{alpha} for the subsequent MCP-1 production. To test the hypothesis, we initiated the culture in the presence of either LPS or IFN-{gamma} alone and then added IFN-{gamma} or LPS, respectively, at later time points (Fig. 5A). Interestingly, the addition of IFN-{gamma} to LPS-activated neutrophils at 3 or 6 h was still effective and showed a tendency to induce higher levels of MCP-1 release than that induced by simultaneous addition of both LPS and IFN-{gamma} at time 0. The addition of IFN-{gamma} was significantly less effective (~50% decrease) when it was added 10 h after the initiation of culture, and only 20% of MCP-1 release was seen when IFN-{gamma} was added after 20 h. In contrast, addition of LPS to cells that were incubated with IFN-{gamma} for 3 h resulted in a significant decrease in MCP-1 release. Only 20–30% of MCP-1 release was seen when LPS was added at 6 h or later time points (Fig. 5A). A similar observation was made using TNF-{alpha} and IFN-{gamma}, with the exception that late addition of IFN-{gamma} was still effective when neutrophils were first activated with TNF-{alpha} (Fig. 5B). These results indicated that initial activation of neutrophils with LPS or TNF was important and the effect of IFN-{gamma} was not due to its priming effect.


Figure 5
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  		FIGURE 5. Initial activation with LPS or TNF-{alpha} is critical for neutrophil MCP-1 release. A, Neutrophils were initially incubated in the presence of either 1 ng/ml LPS or 100 U/ml IFN-{gamma}. Either 100 U/ml IFN-{gamma} or 1 ng/ml LPS was later added to the culture, respectively. Culture supernatants were collected 48 h after the initiation of culture and subjected to ELISA. Data are presented as mean ± SD of four independent experiments with four donors. The value obtained by addition of IFN-{gamma} following initial LPS treatment at each time point was compared with that obtained by addition of LPS following initial IFN-{gamma} treatment. A value of p < 0.05 was considered to be statistically significant. *, p < 0.05; **, p < 0.001, n = 4. The value obtained by addition of LPS following initial IFN-{gamma} treatment at each time point was also compared with that obtained by simultaneous addition of LPS plus IFN-{gamma} at time 0. A value of p < 0.05 was considered to be statistically significant. {dagger}, p < 0.05; {dagger}{dagger}, p < 0.01; {dagger}{dagger}{dagger}, p < 0.001, n = 4. B, Neutrophils were initially incubated in the presence of either 1 ng/ml TNF-{alpha} or 100 U/ml IFN-{gamma}. Either 100 U/ml IFN-{gamma} or 1 ng/ml TNF-{alpha} was later added to the culture, respectively. Culture supernatants were collected 48 h after the initiation of culture and subjected to ELISA. The percentage of MCP-1 release was calculated by comparing the concentration of MCP-1 obtained after each treatment to that obtained by simultaneous addition of LPS and IFN-{gamma} at time 0. Data are presented as mean ± SD of five independent experiments with five donors. The value obtained by addition of IFN-{gamma} following initial TNF-{alpha} treatment at each time point was compared with that obtained by addition of TNF-{alpha} following initial IFN-{gamma} treatment. A value of p < 0.05 was considered to be statistically significant. *, p < 0.05, n = 5. The value obtained by the addition of TNF-{alpha} following initial IFN-{gamma} treatment at each time point was also compared with that obtained by simultaneous addition of TNF-{alpha} plus IFN-{gamma} at time 0. A value of p < 0.05 was considered to be statistically significant. {dagger}, p < 0.05; {dagger}{dagger}, p < 0.01; {dagger}{dagger}{dagger}, p < 0.001, n = 4.

 
Prolonged neutrophil survival is critical for neutrophil MCP-1 release

We and others (23, 24) previously reported that IFN-{gamma} is a potent antiapoptotic agent for neutrophils. Thus, we next hypothesized that IFN-{gamma}-induced prolonged neutrophil survival may be critical for the MCP-1 production that takes place at a late stage of culture. We tested the hypothesis by quantitating the percentage of annexin V–PI–, nonapoptotic neutrophils after a 24-h incubation in different conditions. As shown in Fig. 6, A and B, only 20% of the cells were annexin V–PI– after a 24-h incubation in medium alone. One nanogram of LPS per milliliter or TNF-{alpha} slightly increased the percentage of annexinV–PI– cells (~25%), as previously reported (25, 26). IFN-{gamma} exhibited a more profound antiapoptotic effect than LPS or TNF-{alpha}, and ~40% of cells were still annexinV–PI–. A combination of LPS plus IFN-{gamma} or TNF plus IFN-{gamma} further increased the percentage of annexin V–PI – cells to ~55%. In contrast, when IFN-{gamma} was added to cells previously incubated with LPS for 10 h, the percentage of annexin V–PI– cells was constantly lower. Addition of IFN-{gamma} was still effective when it was added to cells previously activated for 10 h with TNF-{alpha}. There was a strong correlation between the level of MCP-1 production (Fig. 5) and neutrophil survival induced with each culture condition (Fig. 6). These results strongly support the hypothesis that IFN-{gamma} promoted neutrophil MCP-1 production by prolonging its survival.


Figure 6
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  		FIGURE 6. Neutrophil survival is critical for MCP-1 release. Neutrophils were incubated for 24 h in the presence of different stimuli and the percentages of annexin V–PI– cells were evaluated by flow cytometry. A, Representative profiles of three individual experiments are shown. B, The percentage of annexin V–PI– cells was quantified and statistical analysis was performed. A value of p < 0.05 was considered to be statistically significant. n = 3.

 
To obtain additional evidence supporting our hypothesis, we examined whether neutrophils express MCP-1 mRNA after a 16-h incubation with LPS or TNF-{alpha} alone. Because the percentage of annexin–PI – neutrophils in each culture condition varied, we first isolated total RNA from cells incubated in each condition, and the same amounts (10 µg/lane) of total RNA were subjected to Northern blotting. As shown in Fig. 7, a high level of MCP-1 mRNA was detectable in neutrophils incubated for 16 h with LPS or TNF-{alpha} alone, although the level induced with LPS or TNF-{alpha} alone was lower than that induced in combination with IFN-{gamma}. This result further supported our hypothesis that activation of neutrophils with LPS or TNF-{alpha} is sufficient for MCP-1 expression in neutrophils, independently of IFN-{gamma}, but IFN-{gamma}-induced cell survival is critical for the production and release of the protein.


Figure 7
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  		FIGURE 7. MCP-1 mRNA is expressed in neutrophils incubated with LPS or TNF-{alpha} without IFN-{gamma}. Neutrophils were incubated for 24 h in the presence of TNF-{alpha} (1 ng/ml), TNF-{alpha} (1 ng/ml) plus IFN-{gamma} (100 U/ml), LPS (1 ng/ml), or LPS (1 ng/ml) plus IFN-{gamma} (100 U/ml). Expression of MCP-1 mRNA was examined using Northern blot analysis. To indicate equal loading of each RNA sample, the image of the ethidium bromide-stained gel is also presented. Representative data of two independent experiments with similar results are shown.

 
p38 MAPK plays two different roles in neutrophil MCP-1 production

The p38 MAPK pathway is known to positively regulate the expression and subsequent production of MCP-1 in different cell types (27, 28, 29). Therefore, we examined the role of this kinase in neutrophil MCP-1 production using the p38 MAPK inhibitor SB203850. As shown in Fig. 8A, pretreatment of cells with SB203580 significantly inhibited MCP-1 release induced with low concentrations of LPS plus IFN-{gamma}. The effect of this inhibitor became less evident when the LPS concentration was increased to 1 ng/ml. Unexpectedly, inhibition of p38 MAPK dramatically increased MCP-1 production when 10 ng/ml LPS was used. A similar effect was detected when 10 ng/ml TNF-{alpha} was used instead of LPS (data not shown). SB203580 had no effect on MCP-1 release when cells were incubated in the presence of LPS, TNF-{alpha}, or IFN-{gamma} alone (data not shown).


Figure 8
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  		FIGURE 8. SB203580 has dual effects on neutrophil MCP-1 release. A, Neutrophils were preincubated for 30 min with 10 µM SB203580 and then incubated for an additional 48 h in the presence of different doses of LPS plus 100 U/ml IFN-{gamma}. MCP-1 concentrations were measured by ELISA. Data are presented as mean ± SD of three independent experiments with three donors. A value of p < 0.05 was considered to be statistically significant. n = 3. B, Neutrophils were preincubated for 30 min with or without 10 µM SB203580 and then incubated for an additional 24 h in medium alone, with 10 ng/ml LPS plus 100 U/ml IFN-{gamma}, or with 10 ng/ml TNF-{alpha} plus 100 U/ml IFN-{gamma}. The percentage of annexin V–PI– cells was evaluated by flow cytometry, and the percentage of annexin V–PI– cells was quantified. A value of p < 0.05 was considered to be statistically significant; n = 3.

 
Because we have observed a correlation between neutrophil survival and MCP-1 release as demonstrated above and p38 MAPK has been implicated in neutrophil survival (30, 31), we examined whether SB203580 increased MCP-1 release from neutrophils incubated with 10 ng/ml LPS or TNF-{alpha} plus IFN-{gamma} by prolonging their survival. As shown in Fig. 8B, SB203580 significantly increased the percentage of annexin V–PI– neutrophils in the presence of 10 ng/ml LPS or TNF-{alpha} plus IFN-{gamma}; thus, again there was a strong correlation between increased neutrophil survival and MCP-1 release. Taken together, our results indicated that p38 MAPK plays two different roles; activation of this kinase positively regulates neutrophil MCP-1 production when cells are activated with low concentrations of LPS or TNF-{alpha} plus IFN-{gamma}, but it also negatively regulates when cells are activated with high concentrations of LPS or TNF-{alpha} plus IFN-{gamma}, probably by promoting cell death. In contrast to SB203580, the MEK inhibitor PD98059 had no effect on neutrophil MCP-1 production (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we examined whether activation of TLRs, such as TLR4 or TLR2, induces human neutrophils to produce MCP-1. Although LPS (TLR4 ligand), LTA, or Pam3CSK4 (TLR2 ligand), by itself, did not induce significant MCP-1 production, these stimuli, in combination with IFN-{gamma}, induced a high level of MCP-1 production. We then analyzed the mechanisms whereby LPS and IFN-{gamma} cooperate in this process. Initial activation of neutrophils with LPS, but not IFN-{gamma}, was critical and appeared to be sufficient for the expression of MCP-1 mRNA; however, it did not result in MCP-1 production. Interestingly, IFN-{gamma} markedly prolonged the survival of LPS-activated neutrophils, and this IFN-{gamma}-mediated survival enabled them to produce MCP-1. This is the first report demonstrating that there is a direct relationship between prolonged neutrophil survival and neutrophil function and that the production of MCP-1 is a biological consequence of IFN-{gamma}-mediated neutrophil survival.

As indicated above, LPS is a ligand for TLR4, whereas LTA and Pam3CSK4 are ligands for TLR2. Human neutrophils express both receptors on the surface, although the expression of TLR2 appears to be more abundant (14). Sabroe et al. (16) previously examined selective roles for TLR2 and TLR4 in the regulation of neutrophil activation and life span. Activation of TLR4 with LPS and TLR2 with Pam3CSK4 both induced adhesion molecule expression, respiratory burst, and IL-8 generation at a similar level, whereas TLR4 was the principal regulator of neutrophil survival. In our study, all three TLR ligands, in combination with IFN-{gamma}, induced MCP-1 production in neutrophils; however, LPS was a more potent inducer of MCP-1 production than LTA or Pam3CSK4, supporting the previous notion that TLR2 and TLR4 play selective roles in neutrophil functions.

In addition to TLR ligands, TNF-{alpha}, in combination with IFN-{gamma}, also induced MCP-1 production. MCP-1 production induced with LPS plus IFN-{gamma} was partly dependent on TNF-{alpha} produced during the culture. We attempted to detect TNF-{alpha} in the culture supernatants of neutrophils incubated with LPS plus IFN-{gamma} by ELISA. Despite the clear involvement of TNF-{alpha} in neutrophil MCP-1 production as demonstrated using a neutralizing Ab against this cytokine, we did not detect measurable levels of TNF-{alpha} (data not shown). Bennouna et al. (32) recently reported that mouse neutrophils express cell-associated TNF-{alpha} on their surface and activated mouse DC to produce additional TNF-{alpha}. We therefore examined the presence of cell-associated TNF-{alpha} by flow cytometry but we were also not able to detect cell surface TNF-{alpha} by this method using a commercially available Ab (data not shown). Nevertheless, neutrophil-derived TNF-{alpha} clearly played a role in the induction of MCP-1 production, supporting the role for neutrophil-derived TNF-{alpha} in the development of adaptive immune responses.

IFN-{gamma} is a cytokine produced by activated T cells, NK cells, and macrophages and regulates the development of Th1 responses. Interestingly, IFN-{gamma} also acts on neutrophils that are not traditionally considered to be a player in adaptive immune responses. The priming effects of IFN-{gamma} have been well documented (22). A variety of traditional neutrophil functions may be primed, including increased oxidative metabolism, surface receptor expression, and degranulation. We and others previously reported that IFN-{gamma} possesses a potent antiapoptotic activity for neutrophils (23, 24, 26). In the present study, a larger percentage of neutrophils constantly survived in the presence of IFN-{gamma} than LPS or TNF-{alpha}, both of which are probably better known as antiapoptotic agents for neutrophils (23, 26). Despite its potent antiapoptotic activity for neutrophils, the biological consequence of IFN-{gamma}-mediated neutrophil survival remains unclear. In this study, we have demonstrated that IFN-{gamma}-mediated prolonged neutrophil survival resulted in the production of MCP-1, thus providing the first evidence that the antiapoptotic effect of IFN-{gamma} has a biological consequence. In addition to MCP-1, monokine induced by IFN-{gamma}/CXCL9, IFN-{gamma}-inducible protein 10/CXCL10, and IFN-inducible T cell {alpha} chemoattractant/CXCL11, which are ligands of CXCR3 and regulate the recruitment of activated T cells, can also be produced by neutrophils incubated with IFN-{gamma} plus LPS or TNF-{alpha} (19, 33). However, in contrast to MCP-1 whose mRNA expression was not dependent on IFN-{gamma}, the expression of mRNA for these CXCR3 ligands was completely dependent on IFN-{gamma}. Thus, although activation of neutrophils with LPS plus IFN-{gamma} leads to the production of MCP-1 and CXCR3 ligands, the underlying mechanisms regulating the production of MCP-1 and CXCR3 ligands are different.

It was interesting that the initial activation of neutrophils with either LPS or TNF-{alpha}, followed by IFN-{gamma}, was very effective for their MCP-1 production. During inflammatory responses, infiltrating neutrophils are first activated by Ags and/or TNF-{alpha} at inflammatory sites. These cells will then be exposed to IFN-{gamma} that can be released from macrophages, NK cells, or T cells at later time points. It was previously reported that LPS was capable of inducing IFN-{gamma} release from PBMC (34); therefore, IFN-{gamma} can be present during microbial infection. In the case of DTH, TNF-{alpha} was detected at the reaction sites before IFN-{gamma} (35). Thus, sequential activation of neutrophils with LPS or TNF-{alpha} followed by IFN-{gamma} can occur in vivo and the mechanisms we have demonstrated here are biologically relevant.

GM-CSF also possesses a potent antiapoptotic activity for neutrophils and has been shown to up-regulate MCP-1 production in neutrophils (12). We detected significant levels of MCP-1 in the culture supernatants of neutrophils incubated with GM-CSF and confirmed previous results by others. However, the levels of MCP-1 induced with GM-CSF were ~50% of those induced with LPS plus IFN-{gamma}. There was no synergistic effect between GM-CSF and LPS, and GM-CSF failed to rescue LPS-activated neutrophils from cell death (T. Yoshimura, unpublished data). Thus, the effect of IFN-{gamma} is unique and may not be limited to its antiapoptotic effect. Additional studies are necessary to define the exact mechanism by which IFN-{gamma} promotes MCP-1 production.

p38 MAPK plays an important role in the expression of many proinflammatory cytokines and chemokines, including MCP-1 (27, 36). We attempted to evaluate the role of this kinase in neutrophil MCP-1 production. As we expected, an inhibitor of this kinase reduced the production of MCP-1 when cells were incubated with low concentrations of LPS in combination with IFN-{gamma}. To our surprise, the same inhibitor dramatically increased the production of MCP-1 induced with a higher concentration of LPS plus IFN-{gamma}. This increased MCP-1 production strongly correlated with increased survival of activated neutrophils. Our results suggested that when neutrophils were strongly activated with high concentrations of LPS plus IFN-{gamma}, this activation led to neutrophil death through a mechanism involving the activation of p38 MAPK. IFN-{gamma} could not rescue the cells from cell death at those concentrations as previously reported (26), resulting in a lower level of MCP-1 production. p38 MAPK has been a molecular target for the treatments of many human inflammatory diseases and inhibition of this kinase is hoped to provide relief to patients suffering from the diseases (37). In this study, we have demonstrated that inhibition of p38 MAPK can prolong survival of activated neutrophils and promote the production of MCP-1 and potentially other proinflammatory mediators; therefore, inhibition of p38 MAPK may not be beneficial in some cases. p38 MAPK was previously found to play two conflicting roles in spontaneous apoptosis of neutrophils (30, 31). Aoshiba et al. (31) reported that inhibition of p38 MAPK with SB203580 delayed neutrophil apoptosis, whereas Alvarado-Kristensson et al. (30) reported that p38 MAPK constitutes a survival signal. Additional studies are necessary to define the roles of p38 MAPK in neutrophil apoptosis and functions.

Recently, Tsuda et al. (38) reported the presence of three different neutrophil subsets using mice with different susceptibilities to infection by methicillin-resistant Staphylococcus aureus. One of the subsets, termed PMN-II, was characterized by its production of MCP-1 and IL-10. This led us to hypothesize that human neutrophils activated with LPS plus IFN-{gamma} may represent the subset detected in mice and may produce IL-10. However, there was no detectable level of IL-10 in the culture supernatants of neutrophils incubated in the presence of TNF-{alpha} plus IFN-{gamma} or LPS plus IFN-{gamma}. Thus, these human neutrophils are phenotypically different from those found in mice (38).

Prolonged neutrophil survival is generally considered to be harmful. In contrast to circulating neutrophils whose half-life is 6–10 h, inflammatory, tissue-infiltrating neutrophils can live much longer (39, 40). These cells are likely activated in response to proinflammatory signals present in a tissue microenvironment and release mediators destructive to host tissue. However, the precise roles of prolonged neutrophil survival in the development of immune responses remain unclear. In this study, we have demonstrated one potential outcome resulting from prolonged neutrophil survival. Neutrophils activated through TLRs or TNFR can survive in an environment where IFN-{gamma} is present, and they produce a biologically significant level of MCP-1. This MCP-1, along with CXCR3 ligands which are also produced by neutrophils in the same environment, may amplify the development of adaptive immune responses, especially of Th1 responses.


   Acknowledgments
 
We are grateful to Dr. Joost J. Oppenheim for helpful discussion and to Dr. Scott K. Durum for reviewing this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Teizo Yoshimura, Building 560, Room 31-36, National Cancer Institute-Frederick, Frederick, MD 21702. E-mail address: yoshimut{at}mail.nih.gov Back

3 Abbreviations used in this paper: DC, dendritic cell; DTH, delayed-type hypersensitivity reaction; Pam3CSK4, N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Ser-[S]-Lys; LTA, lipoteichoic acid; PI, propidium iodide. Back

Received for publication October 16, 2006. Accepted for publication May 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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