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The Journal of Immunology, 2007, 179, 3570 -3577
Copyright © 2007 by The American Association of Immunologists, Inc.
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Mouse Neutrophils Require JNK2 MAPK for Toxoplasma gondii-Induced IL-12p40 and CCL2/MCP-1 Release1

Woraporn Sukhumavasi, Charlotte E. Egan and Eric Y. Denkers2

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The MAPK family member JNK/stress-activated MAPK (SAPK) is involved in extracellular stress and proinflammatory cytokine responses, including production of cytokines such as IL-12. The JNK1 and 2 isoforms are widely expressed, but JNK3 is largely restricted to tissues of the brain, testis, and heart. In this study, we focus on mouse neutrophils, a cell type in which JNK/SAPK expression and activity has been given little study. We used Western blot analysis to examine expression patterns of JNK/SAPK in wild-type and JNK2–/– polymorphonuclear leukocytes (PMN). Surprisingly, neutrophils displayed a major deficiency in JNK1 expression, in contrast to macrophages that expressed high levels of both JNK1 and JNK2 MAPK. JNK1 expression was steadily reduced during the neutrophil maturation in bone marrow. We used PMN infection with the protozoan parasite Toxoplasma gondii to determine whether neutrophil JNK2 was functional. The parasite induced rapid JNK2 phosphorylation and intracellular FACS staining demonstrated preferential activation in infected neutrophils. Use of JNK2–/– neutrophils revealed that this MAPK family member was required for PMN IL-12p40 and CCL2/MCP-1 production. The chemotactic response displayed a minor JNK2 dependence but phagocytosis and oxidative burst activity did not require this MAPK. These findings are important because they demonstrate 1) a previously unrecognized unusual JNK expression pattern in mouse neutrophils, 2) JNK2 in PMN is activated by Toxoplasma invasion, and 3) a requirement for JNK2 in PMN IL-12p40 and CCL2/MCP-1 production in response to a microbial pathogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutrophils are well known as acute inflammatory cells that rapidly accumulate in high number at sites of infection, largely in response to the chemokine IL-8/CXCL8 in humans and IL-8/CXCL8-like chemokines in mice (1, 2, 3). In the classical view of polymorphonuclear leukocyte (PMN)3 function, these cells exert antimicrobial activity through phagocytosis, granule exocytosis, and production of reactive oxygen intermediates (4). This is followed by neutrophil apoptotic death and phagocytic removal by macrophages. More recently, neutrophil extracellular traps composed of chromatin and granule proteins have been reported to contribute to killing of extracellular bacteria (5, 6).

Although PMN possess an abbreviated life span under steady-state conditions, their half-life is extended by cytokines including TNF-{alpha}, suggesting that the cells can function for extended periods in a proinflammatory milieu (7, 8). Neutrophils themselves also produce proinflammatory cytokines and chemokines that can be released from preformed stores or produced de novo (9, 10, 11, 12, 13). The ability of neutrophils to produce immunoregulatory cytokines such as IL-12 and TNF-{alpha} suggests that the cells play a role in shaping development of the acquired immune response to infection. Recent studies have found Ag-bearing PMN in secondary lymphoid organs that have the potential to influence generation of Th1 lymphocytes through IL-12 release (14). Increasingly, there is evidence for cross-talk between PMN and dendritic cells (15). Neutrophils infected with the protozoan parasite Toxoplasma gondii release TNF-{alpha} which, in turn, activates dendritic cells (9, 16). Similar neutrophil-DC interactions occur in humans, and this cross-talk is mediated by direct cell contact and glycosylation-dependent interaction between neutrophil MAC-1 and DC-SIGN expressed by dendritic cells (17). In addition, DC can acquire Ag from neutrophils through direct physical contact for presentation to T lymphocytes (18). These combined observations strongly argue for an immunoregulatory function for neutrophils, in addition to their well-known microbicidal activity (19).

Neutrophils are a source of IL-12 during infection, but little is known regarding intracellular signaling molecules involved in PMN production of this cytokine. During Toxoplasma infection, the TLR adaptor molecule MyD88 is required for parasite-induced IL-12 in macrophages, DC, and PMN (20). Neutrophils also produce the monocyte chemoattractant CCL2/MCP-1, a response dependent upon MyD88 and TLR2 (21). Members of the MAPK family influence IL-12 production in cells such as macrophages and DC. In particular, p38 and JNK/stress-activated protein kinase (SAPK) promote IL-12 production, although in some cases JNK is reported to negatively regulate IL-12 (22, 23, 24, 25). ERKs 1 and 2 have been implicated in negative regulation of IL-12 synthesis in macrophages (22, 23, 26, 27). However, little is known regarding MAPK in mouse neutrophil function.

In this study, we investigated JNK/SAPK function in murine PMN. Of the three JNK isoforms, JNK1 and JNK2 are widely expressed but JNK3 is limited to tissues of the brain, testis, and heart (22). Unexpectedly, we found that neutrophil JNK expression is largely restricted to JNK2, unlike macrophages which express both JNK1 and JNK2. Using PMN from JNK2–/– mice, we show a major requirement for JNK2 in neutrophil IL-12 and CCL2/MCP-1 production. Chemotaxis was partially dependent upon JNK2. In contrast, no JNK2 requirement for PMN respiratory burst and phagocytic activity was observed. These results reveal novel aspects of MAPK expression in mouse neutrophils, and they show a critical contribution of JNK2 in PMN production of certain chemokines and cytokines during parasite infection.


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

Female C57BL/6 mice 6–8 wk old were purchased from Charles River Breeding Laboratories or Taconic Farms. Homozygous JNK2 knockout mouse (Mapk9tm1Flv) breeding pairs were obtained from The Jackson Laboratory. They were maintained in the transgenic mouse core facility at the College of Veterinary Medicine, Cornell University (Ithaca, NY), an institution accredited by the American Association for Accreditation of Laboratory Animal Care.

Parasites

T. gondii tachyzoites of the RH strain and transgenic yellow fluorescent protein (YFP)-expressing RH tachyzoites (provided by D. Roos, University of Pennsylvania, Philadelphia, PA) were passaged in human foreskin fibroblasts twice a week in medium consisting of DMEM (Mediatech) supplemented with 1% bovine growth serum (HyClone), 100 U/ml penicillin, and 100 µg/ml streptomycin (both from Invitrogen Life Technologies). Tachyzoites freshly egressed from fibroblasts were washed with PBS and resuspended in complete DMEM (cDMEM), consisting of 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, 30 mM HEPES (all purchased from Invitrogen Life Technologies), 10% bovine growth serum, and 0.05 mM 2-ME in DMEM.

Mouse neutrophil isolation

To obtain neutrophils, mice were i.p. injected with 10% thioglycolate (BD Microbiology Systems), then peritoneal exudate cells (PEC; consisting of 60–70% PMN) were collected 18 h later. Cells were washed and resuspended in PBS. Neutrophils were purified from PEC by continuous Percoll gradient centrifugation. One hundred percent Percoll (Amersham Biosciences) was adjusted to pH 7.4 with 10 M HCl. The PEC preparation was mixed with the pH-adjusted Percoll at a ratio 1:9, then the mixture was transferred to a 10-ml polycarbonate tube (Beckman Coulter). Ultracentrifugation was performed at 60,650 x g for 65 min at 4°C with a 50 Ti rotor (Beckman Coulter). The neutrophil-enriched layer was harvested using a gel-loading pipette tip. Cells were washed with PBS and resuspended in cDMEM. More than 90% PMN purity was routinely obtained, as determined by morphology after differential staining (Fisher Scientific). PMN viability, monitored by trypan blue exclusion was routinely >98%. Cells were counted at least three times to ensure accurate quantitation.

Magnetic labeling of bone marrow cells and cell sorting

Mouse bone marrow single-cell suspension was prepared followed by magnetic labeling with CD11b MicroBeads (Miltenyi Biotec). Positive selection for CD11b+ cells was performed twice by an AutoMACS Separator according to the manufacturer’s instruction (Miltenyi Biotec). Enriched-CD11b+ bone marrow cells were then stained with PerCP-Cy5.5-conjugated anti-Gr-1 mAb and PE-conjugated anti-CD11b mAb (BD Pharmingen). A FACSAria fluorescence-activated cell sorter (BD Biosciences) was used to isolate the bone marrow CD11b+ cells into three different populations based on the surface expression level of CD11b and Gr-1: CD11bintGr-1int; CD11blowGr-1high; and CD11bhighGr-1high.

Mouse bone marrow-derived macrophage preparation

Macrophages were generated from mouse bone marrow in L929 supernatant containing M-CSF as described previously (28).

Flow cytometry

For intracellular staining of phosphorylated JNK, YFP-expressing tachyzoites were incubated with PEC (15:1 ratio of parasites to cells). At various times, cells were transferred to a 96-well plate and washed with ice-cold PBS containing 1% bovine growth serum and 0.01% sodium azide (FACS buffer). Samples were fixed with 3% paraformaldehyde for 15 min followed by incubation with anti-CD16/32 (2.4G2) to block nonspecific Ab binding to Fc{gamma}RIII/II. Surface staining for Gr-1 was performed for 15 min with PerCP-Cy5.5-conjugated anti-Gr-1 mAb (BD Pharmingen). For phenotypic analysis of purified neutrophils, cells were costained with the following PE-conjugated Ab: anti-F4/80 (Invitrogen Life Technologies), anti-CD11c (BD Biosciences), anti-CD11b (BD Biosciences), anti-MHC class II (eBioscience), anti-CD62L (eBioscience), anti-CD80 (eBioscience), and anti-CD86 (BD Biosciences). For intracellular staining of phospho-JNK, cells were permeabilized with 0.075% saponin in PBS for 30 min followed by blocking with FACS buffer for 10 min. Intracellular staining was accomplished with Alexa Fluor 647-conjugated phospho-SAPK/JNK (T183/Y185) mouse mAb (Cell Signaling Technology) for 30 min at room temperature. Samples were acquired on a FACSCalibur (BD Biosciences) cytometer collecting 10,000 Gr-1+ events. Data were analyzed using FlowJo software (Tree Star).

Immunoblot analysis

PMN and tachyzoites were added to a 24-well plate and infection was synchronized by brief centrifugation (550 x g, 3 min, 4°C). After incubation for various times (37°C, 5% CO2), supernatants were removed and cell lysates were prepared for immunoblot analysis. Bone marrow-derived macrophages were used as a control for JNK activation by incubation for 20–30 min with 100 ng/ml LPS (Escherichia coli strain 055:B5; Sigma-Aldrich).

For immunoblot analysis, samples were lysed with SDS-PAGE reducing sample buffer and passed through a 27-gauge needle three times to reduce sample viscosity. Lysates were boiled for 5 min, run on a 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane. Membranes were blocked with 3% nonfat dry milk (Nestle) in PBS for 20 min. They were probed with a rabbit anti-total JNK polyclonal Ab (Upstate Cell Signaling Solutions) or anti-phospho-JNK according to the manufacturer’s recommendation (Cell Signaling Technology). HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology) was used as a secondary Ab. Bands were visualized by developing with luminol chemiluminescent substrate (Cell Signaling Technology) and exposure to autoradiographic film.

Cytokine/chemokine ELISA

For cytokine protein measurement, neutrophils from WT and JNK2–/– mice were resuspended in cDMEM and plated in a 96-well plate. Tachyzoites were added and infection was synchronized by brief centrifugation. The cells were incubated at 37°C in humidified 5% CO2 for various times, then supernatants were recovered for ELISA. Production of IL-12p40 and CCL2/MCP-1 was measured by ELISA as previously described (21).

Multiplex bead immunoassay

Neutrophil supernatants from infected cultures were subjected to Multiplex bead immunoassay according to the manufacturer’s instruction (BioSource International). The following 19 mouse cytokines and chemokines were measured using a Luminex 100 instrument (Qiagen): IL-12p40/p70, CCL2/MCP-1, GM-CSF, IFN-{gamma}, IL-1{alpha}, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-{gamma}-inducible protein 10, KC, MIG, MIP-1{alpha}, TNF-{alpha}, and vascular endothelial growth factor (VEGF).

Oxidative burst assay

PMN were stimulated with PMA (200 ng/ml) and fMLP (10–6 M) (EMD Biosciences) in the presence of 0.2% NBT (Sigma-Aldrich). After 30 min (37°C, 5% CO2), cells were solubilized with 10% SDS and 0.1 N HCl, then sample OD (570 nm) were measured on a spectrophotometer.

Phagocytosis assay

PMN were incubated (1 h, 37°C, 5% CO2) with 1 µm of fluorescent yellow-green latex beads (Sigma-Aldrich) that had been preopsonized with normal mouse serum for 1 h. Internalization of beads was determined by flow cytometry using a FACSCalibur (BD Biosciences) flow cytometer and data were analyzed with FlowJo software (Tree Star).

Chemotaxis assay

Assays for chemotaxis used a 96-well chemotaxis chamber (3-µm pore size; NeuroProbe). The assay was set up as described previously (16) using 45,000 neutrophils. The chemoattractants fMLP (10–6 M) and KC (100 ng/ml; AbD Serotec) were used in the study. The assay was terminated after 45 min at 37°C, then the number of migrated cells was enumerated by microscopy.

Statistics

The difference of means was compared using Student’s t test with p < 0.05 considered statistically significant. All experiments were repeated at least two times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Isolation of mouse peritoneal neutrophils

To obtain purified neutrophils for in vitro studies, we elicited peritoneal cell recruitment by i.p. injection of 10% thioglycolate and collected exudate cells 18 h later. PEC were routinely composed of 60–70% neutrophils and 20–25% macrophages, with small numbers of eosinophils, mast cells, and lymphocytes (Fig. 1A). To purify neutrophils, PEC were subjected to ultracentrifugation over a Percoll continuous density gradient. This procedure resulted in cell preparations composed of >90% neutrophils (Fig. 1B). We performed flow cytometry to further assess the phenotype of the Gr-1+ neutrophil population. As shown in Fig. 1C, the cells expressed high levels of CD11b and CD62L, but lacked expression of CD11c and MHC class II. The neutrophil population was also negative for F4/80 (data not shown). Interestingly, the cells expressed intermediate levels of costimulatory molecule CD86 and low levels of CD80. Although we are uncertain as to the functional significance of this result, expression of costimulatory molecules by neutrophils isolated from humans has also been reported (29, 30).


Figure 1
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  		FIGURE 1. Mouse neutrophils from thioglycolate-elicited peritoneal cells before and after purification. A, PEC were obtained by peritoneal lavage with ice-cold PBS 18 h after i.p. thioglycolate injection. The cells were comprised of 60–70% PMN, 20–25% macrophages (M{phi}), low numbers of eosinophils (eos), mast cells (mast), and lymphocytes (data not shown). B, PEC were subjected to PMN purification over a continuous Percoll gradient immediately following harvest. More than 90% neutrophil purity was routinely obtained. C, Purified neutrophils were stained with anti-Gr-1 Ab and a panel of cell surface markers. Expression of CD11b, CD11c, CD62L, CD80, CD86, and MHC class II (MHCII; thick black line in each histogram) is shown on the gated Gr-1+ population relative to staining with isotype controls (thin gray lines in each histogram).

 
Diminished expression of JNK1 in mouse neutrophils

The MAPK family members are implicated in proinflammatory cytokine responses, and we previously reported a requirement for JNK in macrophage IL-12 production (31). Because neutrophils produce IL-12 during microbial stimulation, we assessed the status of JNK1/2 in these cells. Fig. 2 shows levels of total JNK in macrophages compared with purified neutrophils. As expected, WT macrophages expressed high levels of 46- and 54-kDa proteins corresponding to JNK1 and JNK2, respectively. In marked contrast, neutrophils were almost completely devoid of JNK1. JNK3 migrated with a mass of 54 kDa and therefore comigrated with JNK2. However, using neutrophils from JNK2–/– mice, we established that the 54-kDa band corresponded to JNK2 protein (Fig. 2).


Figure 2
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  		FIGURE 2. JNK2 is the major JNK/SAPK isoform expressed in mouse neutrophils. Total cell lysates of bone marrow-derived macrophage (M{phi}) and highly purified PMN from WT or JNK2–/– mice were subjected to immunoblot analysis using a JNK-specific polyclonal Ab and an actin-specific Ab as a protein loading control. The amount of macrophage and PMN protein loaded was 5, 10, and 15 µg/lane. Positions of JNK1 and 2 isoforms are marked by arrows. This experiment was repeated twice with similar result. MW, Molecular weight marker.

 
Down-regulation of JNK1 during neutrophil maturation

We next sought to determine where JNK1 expression was down-modulated during neutrophil differentiation in the bone marrow. PMN and their precursors in bone marrow can be distinguished by morphology and expression of surface markers Gr-1 and CD11b (32). Accordingly, we enriched for CD11b+ bone marrow cells using immunomagnetic beads, then sorted cells into three different populations consisting of CD11bintGr-1int, CD11blowGr-1high, and CD11bhighGr-1high populations (Fig. 3A, a–c, respectively). Each population was subjected to Diff-Quick staining (Fig. 3B). In agreement with previous results (32), the morphology of CD11bintGr-1int cells was consistent with promyelocyte/myelocyte stage cells (Fig. 3Ba), whereas the CD11blowGr-1high and CD11bhighGr-1high displayed morphology consistent with immature PMN and multilobular nucleated mature PMN (Fig. 3B, b and c, respectively). To examine the expression levels of JNK1 in each population, Western blot analysis was performed on protein lysates from the sorted bone marrow cell populations, as well as on purified peritoneal neutrophils from WT and JNK2–/– mice and bone marrow-derived macrophages for comparison. Fig. 3C shows a steady decline in JNK1 and JNK2 expression associated with neutrophil maturation. However, based on densitometric analysis, the decrease in JNK1 was more drastic. Thus, the JNK2:JNK1 ratio in promyelocyte/myelocytes, immature PMN, and mature PMN was 1.9, 3.7, and 7.3, respectively. In elicited neutrophils obtained from the peritoneal cavity, the JNK2:JNK1 ratio was 15.8. In contrast, JNK1 was expressed at a higher level in macrophages (JNK2:JNK1 = 1.6) and CD11b– bone marrow cells (JNK2:JNK1 = 1.7) (Fig. 3C).


Figure 3
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  		FIGURE 3. JNK expression during neutrophil maturation. A, CD11b+ bone marrow cells were enriched by magnetic sorting, then further stained and FACS sorted based on the expression level of CD11b and Gr-1. The gated populations define: a, promyelocytes/myelocytes; b, immature neutrophils; and c, mature neutrophils. B, Sorted populations a–c were subjected to cytospin and Diff-Quick staining to determine the cell morphology. C, Bone marrow (BM) CD11b– cells as well as populations a–c were subjected to Western blot analysis probing with JNK polyclonal Ab. These were compared with lysates of bone marrow-derived macrophages (M{phi}) and WT and JNK2–/– neutrophils isolated from the peritoneal cavity after thioglycolate elicitation. The same membrane was later stripped and reprobed with actin Ab. Data shown are representative of two independent experiments.

 
Neutrophil JNK2 is activated during microbial infection

We used T. gondii, an intracellular pathogen that activates JNK during macrophage infection (31, 33), to examine whether neutrophil JNK2 undergoes similar activation. Purified PMN were incubated with tachyzoites, and intracellular tachyzoites (an average of one per cell) were subsequently observed within 20 min of infection (Fig. 4A). At 18 h postinfection, intact intracellular tachyzoites were apparent, although rarely more than four per cell (Fig. 4B). Absence of any overt signs of parasite death or degradation suggests that live tachyzoites actively invade PMN rather than being internalized by neutrophil phagocytosis. During infection of PMN, the parasite triggered JNK2 phosphorylation and the response was maximal between 20 and 30 min (Fig. 4C). This result correlates with the kinetics of T. gondii-mediated JNK activation in infected bone marrow-derived macrophages (28).


Figure 4
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  		FIGURE 4. T. gondii infection and activation of JNK in mouse neutrophils. Neutrophils were infected with tachyzoites and cells were examined 20 min (A) and 18 h postinfection (B). C, PMN were stimulated with tachyzoites for the period of time indicated. Cell lysates were subjected to immunoblot analysis probing with polyclonal Ab specific for phospho-JNK (p-JNK). The membrane was then stripped and reprobed with total JNK Ab. LPS-stimulated bone marrow-derived macrophages were used as a positive control for JNK activation. Data shown are representative of three independent experiments.

 
Rapid JNK activation in T. gondii-infected, but not bystander PMN

Phosphorylation of neutrophil JNK2 could be induced by secreted products of extracellular tachyzoites, by products released during or shortly after invasion, or the combination of the two. To address this issue, we used YFP-expressing T. gondii to infect PEC that were subsequently stained with anti-Gr-1 Ab. This enabled us to distinguish infected and noninfected Gr-1+ populations (Fig. 5, A and B). To examine the JNK2 phosphorylation in each population, intracellular phospho-specific JNK staining was also performed. The intensity of phosphorylation between infected and noninfected Gr-1+ PMN (thick lined-boxes in Fig. 5B) was compared with Gr-1+ neutrophils unexposed to parasites (thin-lined box in Fig. 5A). Interestingly, JNK phosphorylation occurred predominantly in infected neutrophils, with little or no activation in the bystander noninfected population (Fig. 5C). In this situation, activation occurred with rapid kinetics, reaching maximal levels at 20 min, then shifting back to basal level by 30 min postinfection.


Figure 5
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  		FIGURE 5. JNK activation occurs predominantly in T. gondii-infected-Gr-1+ PMN. A and B, Use of YFP-expressing tachyzoites allows identification of infected and noninfected PMN. Thioglycolate-elicited PEC were stained with anti-Gr-1 Ab in the absence of tachyzoite (TZ) infection (A). Alternatively, cells were infected, and flow cytometric analysis was performed 20 min postinfection (B). C, infected (rectangle in B) and noninfected (square in B) neutrophils were subjected to phospho-specific JNK staining and analyzed by flow cytometry at the indicated time points (min) after in vitro infection. The thick lines show phospho-JNK staining in infected and noninfected populations compared with phospho-JNK staining in PMN not exposed to parasites (thin line in C and area defined in A). This experiment was repeated three times with equivalent results.

 
T. gondii-induced IL-12p40 and CCL2/MCP-1 production in mouse PMN requires JNK2

Previously, we found that PMN produce both IL-12p40 and CCL2/MCP-1 in response to Toxoplasma (21). Therefore, we examined the role of JNK2 in production of these mediators of inflammation. Neutrophils from WT and JNK2–/– mice were incubated with T. gondii tachyzoites at a multiplicity of infection of 0.5:1 and supernatants were subsequently collected for ELISA. In WT PMN, IL-12p40 production was detectable at 4 h, then peaked and maintained at ~1.6 ng/ml for up to 21 h (Fig. 6A). The kinetics of CCL2 production differed, in that this chemokine was not detected until 8 h postinfection and its production increased steadily throughout the time course (Fig. 6B). These results are consistent with our previous data suggesting that IL-12p40 and CCL2/MCP-1 production during T. gondii infection are independently regulated (21). In sharp contrast to the behavior of WT neutrophils, JNK2–/– PMN failed to produce detectable levels of either IL-12p40 or CCL2/MCP-1 (Fig. 6). We extended these studies by using a Multiplex Bead Immunoassay to assay neutrophil supernatants for 19 cytokines and chemokines (listed in Materials and Methods). In this assay, we confirmed that Toxoplasma stimulates JNK2-dependent IL-12 and CCL2/MCP-1. In addition, we found that the neutrophils release MIP-1{alpha} and VEGF in a manner largely independent of infection and, more importantly, without a requirement for JNK2 (Fig. 6C).


Figure 6
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  		FIGURE 6. JNK2 is required for T. gondii-mediated IL-12p40 and CCL2/MCP-1 production. Thioglycolate-elicited, Percoll gradient-purified neutrophils from WT and JNK2–/– mice were incubated with T. gondii at a multiplicity of infection of 0.5:1. At the indicated time points, supernatants were collected for measurement of IL-12p40 (A) and CCL2/MCP-1 (B) production by ELISA. C, Of 19 cytokines and chemokines measured from the supernatants by Multiplex Bead Immunoassay, IL-12p40, CCL2/MCP-1, MIP-1{alpha}, and VEGF were detected. Med, Levels of cytokine/chemokine in neutrophil supernatants after culture for 21 h in medium alone. Data shown are representative of three independent experiments.

 
Neutrophil respiratory burst, phagocytosis, and chemotaxis in the absence of JNK2

In addition to the role of JNK2 in IL-12p40 and CCL2/MCP-1 production, we asked whether this MAPK was involved in other neutrophil functions. To address this question, we performed three assays of PMN function, namely, oxidative burst activity, phagocytosis, and chemotaxis. First as shown in Fig. 7A, both WT and JNK2–/– neutrophils produced an equivalent oxidative burst in response to PMA and fMLP. Second, we determined the ability of WT and JNK2–/– PMN to phagocytose opsonized 1-µm fluorescent beads. As shown in Fig. 7B, WT and JNK2–/– neutrophils were able to phagocytose the beads to the same extent (WT neutrophils, 78% bead positive; JNK2–/– neutrophils, 82% bead positive). In these experiments, we confirmed by microscopy that cell-associated fluorescence reflected internalization of microbeads rather than simple adsorption to the neutrophil surface (Fig. 7B, inset). Third, we used fMLP and the IL-8-related chemokine KC to induce PMN chemotaxis across a Transwell membrane. As shown in Fig. 7C, there was a minor decrease in fMLP-stimulated transmigration in the absence of JNK2 that was not statistically significant (p = 0.07). However, although KC induced weaker chemotaxis, the response was reduced by ~50% in the absence of JNK2 (p < 0.01). In sum, we could not detect a requirement for JNK2 in neutrophil phagocytosis or oxidative burst, but there was a partial requirement for this MAPK molecule in neutrophil chemotaxis.


Figure 7
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  		FIGURE 7. Neutrophil oxidative burst, phagocytosis, and chemotaxis proceed normally in the absence of JNK2. A, Oxidative burst. PMN were stimulated with PMA (0.2 µg/ml) and fMLP (10–6 M) along with NBT solution for 20 min at 37°C. Cells were solubilized in 10% SDS/0.1 N HCl. OD was measured at 570 nm. B, Phagocytosis. PMN were incubated 1 h with 1 µm of green fluorescent beads precoated with normal mouse serum. FACS analysis was used to examine phagocytosis of beads by PMN. Inset, bead-phagocytosed PMN. C, Chemotaxis. PMN were cultured in a 3-µm pore Transwell membrane in contact with fMLP (10–6 M) and KC (100 ng/ml) in the lower chamber. Number of migrated cells was determined after 40 min by counting under the microscope at least three different high-power fields (HPF). *, p = 0.07 and **, p < 0.01 (WT vs JNK2 knockout). Each of these experiments was performed twice with the same result.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results of this study demonstrate that expression of JNK MAPK molecules in mouse PMN is almost completely restricted to JNK2. This contrasts with macrophages, which express both JNK1 and JNK2. We were able to trace down-regulation of JNK1 protein during development of neutrophils through promyelocyte/myelocyte stages to immature neutrophils to mature neutrophils in the bone marrow. Infection of PMN with T. gondii resulted in JNK2 phosphorylation in a manner kinetically similar to that occurring in macrophages. Using PMN isolated from JNK2–/– mice, which are devoid of all JNK isoforms, we demonstrated a requirement for JNK2 in infection-induced IL-12p40 and CCL2/MCP-1 production. Nevertheless, production of MIP-1{alpha} and VEGF was normal in the absence of JNK2. Other neutrophil functions, namely, phagocytosis and respiratory burst activity, occurred in the absence of JNK2. We detected a partial JNK2 requirement in the neutrophil chemotactic response and indeed others have also found involvement of JNK in neutrophil chemotaxis (34, 35).

We previously reported that Toxoplasma induces both IL-12p40 and CCL2 in mouse neutrophils (21, 36). The importance of IL-12 during infection with this opportunistic pathogen has long been recognized (37, 38). Absence of IL-12 results in uncontrolled parasite proliferation and death of the host. More recent data suggest that this is the result of Th1 cell secretion of IL-10, rather than complete failure to generate IFN-{gamma}-positive Th1 T lymphocytes as originally proposed (39). The role of CCL2 during T. gondii infection is currently under investigation. However, the chemokine appears to be involved in recruiting a novel population of Gr-1+ monocytes capable of microbicidal activity against Toxoplasma and other intracellular pathogens (40, 41). The importance of these cells is suggested by the finding that mice lacking CCR2, the receptor for CCL2, are susceptible to T. gondii (40).

In the absence of an Ab capable of specifically depleting neutrophils in vivo or neutrophil-specific cytokine/chemokine knockout mice, it is difficult to directly address the role of neutrophil-derived cytokines and chemokines such as IL-12 and CCL2 in the innate response to infection. Nevertheless, PMN accumulate at high numbers at sites of infection and in secondary lymphoid organs during the early response to Toxoplasma (42). Given the fact that neutrophils produce a large battery of cytokines and chemokines during in vitro stimulation with Toxoplasma and other stimuli (11, 13), it seems likely that production of these mediators plays a role in the outcome of infection in vivo, either through direct effects on T lymphocytes or by influencing other immune effectors such as DC (16, 17, 18).

This study is the first to demonstrate a requirement for JNK in mouse neutrophil IL-12p40 and CCL2 production. Previously, we reported that neutrophil production of both mediators, like production of IL-12 by DC and macrophages, is dependent upon TLR adaptor molecule MyD88 (20, 21). We found that TLR2 was required for CCL2 induction and indeed others have reported that absence of this TLR increases susceptibility to T. gondii (21, 43). Interestingly, neutrophil IL-12 did not require TLR2. It is possible that TLR11, recently identified as a mouse receptor for parasite profilin involved in DCl IL-12 production (44), also drives neutrophil IL-12 release. Further evidence that regulation of IL-12 and CCL2 differs in neutrophils comes from the combined findings that IL-10 down-regulates IL-12 but not CCL2 and an IFN-{gamma}-STAT1 pathway induces CCL2 but not IL-12p40 secretion (21). In the present study, circumstantial evidence for independent regulation comes from the disparate kinetics of IL-12 and CCL2 release during neutrophil infection (Fig. 6).

Although we did not examine responses of human neutrophils, other studies suggest that JNK/SAPK is regulated differently than in mouse PMN. Several reports indicate the presence of JNK1 in human neutrophils, although it has also been reported that JNK2 is the dominant isoform in these cells (45, 46, 47, 48, 49). More interestingly, it has been reported that inflammatory cytokine production in human PMN is independent of JNK and the downstream transcription factor complex AP-1 (50). Although this particular study did not specifically examine either IL-12p40 or CCL2, a variety of PMN chemical activation stimuli failed to induce nuclear translocation of JNK or activation of downstream Jun/Fos proteins. Nevertheless, we note that another group reported a JNK requirement for CCL2 production by human PMN (46), a result in line with our findings here.

In several infection models, there is evidence that JNK1 and JNK2 fulfill distinct functions in the immune response. For example, JNK1 is involved in activated T cell survival during infection with lymphocytic choriomeningitis virus, whereas JNK2 controls proliferation of virus-specific CD8+ T lymphocytes (51, 52). The concept that there are noncompensatory functions for JNK1 and JNK2 is also suggested by reports that Th1 effector cell generation is defective in the absence of JNK2 and that lack of JNK1 confers susceptibility to Leishmania major (53, 54). Interestingly, a recent report implicated JNK2 in the response to Plasmodium falciparum glycosylphosphatidylinositol and pathogenesis of cerebral malaria (55). There is also evidence for complex interplay between JNK1 and JNK2 insofar as JNK2 has been reported to bind to c-Jun, inducing its degradation, whereas JNK1 functions as the major c-Jun kinase following cell stimulation (56). Collectively, these studies indicate nonredundant functional roles for JNK molecules in the immune system.

In the absence of an ability to artificially enforce JNK1 expression in neutrophils, it is difficult to determine why this MAPK family member is down-regulated during the neutrophil differentiation program. Neutrophils are well known to be preprogrammed for accelerated apoptotic cell death, a response largely unique to this cell type. Although some studies indicate a role for JNK in promoting programmed cell death (57, 58), others indicate that JNK signaling suppresses apoptosis (59, 60, 61). Therefore, it is possible that down-modulation of JNK1 could be involved in promoting an apoptotic death program in neutrophils.


   Acknowledgments
 
We thank A. Bierly for critical review of this manuscript, E. Rhoades and C. Streeter for assistance with the multiplex assay, and M. Hossain for expert technical assistance.


   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 work was supported by National Institutes of Health Grant AI47888 (to E.Y.D.) and a fellowship from the King Anandamahidol Foundation (to W.S.). Back

2 Address correspondence and reprint requests to Dr. Eric Denkers, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address: eyd1{at}cornell.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; DC, dendritic cell; SAPK, stress-activated protein kinase; YFP, yellow fluorescent protein; PEC, peritoneal exudate cell; WT, wild type; VEGF, vascular endothelial growth factor. Back

Received for publication March 2, 2007. Accepted for publication July 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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