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Originally published online as doi:10.1189/jlb.0606399 on November 9, 2006

Published online before print November 9, 2006
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(Journal of Leukocyte Biology. 2007;81:412-420.)
© 2007 by Society for Leukocyte Biology

Infiltrating neutrophils induce allospecific CTL in response to immunization with apoptotic cells via MCP-1 production

Yoshiko Shiratsuchi, Takuya Iyoda, Naoko Tanimoto, Daisuke Kegai, Kisaburo Nagata and Yoshiro Kobayashi1

Division of Molecular Medicine, Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Japan

1 Correspondence: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. E-mail: yoshiro{at}biomol.sci.toho-u.ac.jp

ABSTRACT

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity. As neutrophils are known to function as effectors as well as regulators in the immune response, we examined the roles of infiltrating neutrophils in alloantigen-specific CTL induction after immunization with late apoptotic P388 cells. The CTL induction and infiltration of CD8+ T cells into the peritoneal cavity were inhibited by depletion of neutrophils by anti-Gr-1 mAb or inhibition of neutrophil infiltration by anti-MIP-2 antibody, suggesting that neutrophils are involved in CD8+ T cell infiltration into the peritoneal cavity. It is known that MIP-1{alpha}, MIP-1ß, and MCP-1 are capable of attracting CD8+ T cells and that they are produced by neutrophils. These chemokines were detected in the peritoneal cavity, and among them, MCP-1 production was reduced remarkably by suppression of neutrophil infiltration. Moreover, infiltration of CD8+ T cells into the peritoneal cavity as well as CTL activity was clearly reduced by administering anti-MCP-1 antibody i.p. Furthermore, the CTL induction and infiltration of CD8+ T cells in neutrophil-depleted mice were restored significantly by administering recombinant murine MCP-1 into the peritoneal cavity. These results indicate that MCP-1 appears to link infiltration of neutrophils with CTL induction.

Key Words: peritoneal cavity • inflammation • chemokines • immune response

INTRODUCTION

Neutrophils are the earliest cells to arrive at the inflammatory site of an infection and serve as the first line of host defense against infections by ingesting and killing bacteria [1 , 2 ]. Neutrophils also play an important role in promoting [3 4 5 6 ] or suppressing [7 ] the Th1 immune response, which is mediated partly by induction of cytokines or chemokines.

MIP-1{alpha}, MIP-1ß, and MCP-1 are recently reported to serve as chemoattractants for Th1 cells [8 , 9 ]. MIP-1{alpha} and MCP-1 are also reported to enhance antigen-specific CTL induction [10 11 12 13 ]. Moreover, several studies revealed that these three chemokines are also secreted by neutrophils [14 15 16 ]. Indeed, one study revealed that MIP-1{alpha}/ß released from neutrophils are involved in recruitment of macrophages to inflammatory sites [14 ].

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells, a murine leukemic cell line, or late apoptotic CTLL-2 cells, an IL-2-dependent cytotoxic T cell line, caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity [17 18 19 ]. The infiltration of neutrophils was caused by MIP-2, a murine homologue of IL-8 [18 , 19 ].

In the present study, we examine whether the infiltrating neutrophils into the peritoneal cavity are involved in alloantigen-specific CTL induction after immunization with late apoptotic P388 cells. Then we examine the role of neutrophils in the production of MIP-1{alpha}, MIP-1ß, or MCP-1 in the peritoneal cavity and finally, examine whether the chemokine(s) play a role in recruiting CD8+ CTLs into the peritoneal cavity.

MATERIALS AND METHODS

Mice
C57BL/6 mice (6–8 weeks, male) were purchased from SLC (Shizuoka, Japan) and were maintained in a specific, pathogen-free facility in our university.

Cell preparation
DBA/2 mouse (H-2d)-derived leukemic P388 cells were maintained in RPMI-1640 medium containing 7% FCS (Gibco-BRL, Gaithersburg, MD) and 1 x 10–5 M 2-ME. C57BL/6 mouse (H-2b)-derived lymphoma EL4 cells and BALB/c (H-2d) mouse-derived B cell lymphoma A20.2J cells were maintained in RPMI-1640 medium containing 7% FCS. One day before induction of apoptosis, the cell viability of P388 cells was nearly 100%, as confirmed with Trypan blue dye exclusion test. To induce apoptosis, the cell density of P388 cells was adjusted to 5 x 105 cells/ml in culture medium, followed by incubation at 37°C for 24 h in the presence of etoposide (Wako, Osaka, Japan) at the final concentration of 1 µg/ml. The apoptotic P388 cells were at a late stage, as assessed as to DNA ladder formation and staining with propidium iodide [18 ]. The cells were then washed with PBS three times, and they (2x107 cells) were injected into the peritoneal cavity of C57BL/6 mice (H-2b) three times every 4 days. Necrotic P388 cells (2x107 cells) were obtained by one cycle of freeze-thaw, and complete necrosis was confirmed with Trypan blue dye exclusion test.

Cytotoxicity assays
Five days after the last immunization, the cytotoxic activity in the peritoneal exudate cells (PEC) or splenocytes was examined by a standard 51Cr release assay. For the assay, 51Cr-labeled P388 cells, A20.2J cells, or EL4 cells were used as target cells. After a 4-h incubation, supernatants were harvested and counted for the radioactivity with a {gamma} counter. The percentage of specific 51Cr release was defined as (experimental cpm–spontaneous cpm)/(maximum cpm–spontaneous cpm) x 100%. Here, the spontaneous release and the maximum release represent 51Cr release from target cells in medium alone and that from target cells lysed with 1N NaOH, respectively. All the assays were performed in triplicate.

Flow cytometric analysis
Infiltrated cells into the peritoneal cavity (5x105) were pretreated with 0.5 µg Fc block (anti-Fc{gamma}RIII/II mAb prepared from the supernatant of 2.4G2 hybridoma cells) and 0.5 µg mouse IgG1, prepared from the ascites fluid of myeloma MOPC-21 cells for 30 min on ice, followed by staining with FITC-conjugated anti-Gr-1 mAb (mouse neutrophil marker; Clone RB6-8C5, provided by Dr. Fujiro Sendo, Yamagata University, Japan), FITC-conjugated anti-CD11b (membrane-activated complex-1{alpha} chain) mAb (Clone M1/70, American Type Culture Collection, Manassas, VA), FITC-conjugated anti-CD8{alpha} (Ly2) mAb (BD Biosciences, San Jose, CA), or FITC-conjugated isotype control mAb (BD Biosciences) and subsequent incubation for 30 min on ice. Except for several mAb from BD Biosciences, the mAb were conjugated with FITC in our laboratory according to the standard method. After washing with PBS containing 2% FCS twice, the cells were analyzed by flow cytometry with a FACScan (BD Biosciences) using CellQuest software.

Neutrophil depletion
For neutrophil depletion, we used antimouse granulocyte antibody (anti-Gr-1 mAb) against Ly-6G, an antigen on the surface of granulocytes in murine bone marrow [20 ]. A total of 200 µg mAb was administered i.p., 1 day before injection of apoptotic P388 cells. Rat anti-HLA mAb, which was prepared with Clone SFR8-B6, similarly as described above, was injected as a control.

Inhibition of neutrophil infiltration
To suppress neutrophil infiltration, we used anti-MIP-2 polyclonal antibody (pAb) [19 ]. A total of 800 µg anti-MIP-2 pAb was administered i.p., just before injection of apoptotic P388 cells. Although keratinocyte-derived chemokine (KC) is also known to be a chemoattractant for mouse neutrophils, the anti-MIP-2 pAb does not bind to KC [19 ]. Anti-GST pAb, which was prepared similarly as described above, was injected as a control.

Measurements of chemokines
To measure the protein level of MIP-1{alpha}, MIP-1ß, or MCP-1 in the peritoneal cavity, the supernatants of peritoneal lavage fluid were harvested at various times after the third injection of apoptotic P388 cells. The mouse MIP-1ß and MCP-1 levels were determined using a DuoSet ELISA development system (R&D Systems, Minneapolis, MN) and mouse MIP-1{alpha} using an ELISA development kit (PeproTech, London, UK). The detection limits for MIP-1{alpha}, MIP-1ß, and MCP-1 were 8 pg/ml, 15.6 pg/ml, and 3.9 pg/ml, respectively.

Preparation of pAb against MCP-1
The plasmid for expression of GST-MCP-1 fusion protein was constructed by inserting the fragment of the MCP-1 cDNA encoding the open reading frame into GST fusion expression vector pGEX-6P3. The GST-MCP-1 fusion protein was expressed in Escherichia coli BL-21 in the presence of 1 mM isopropyl-1-thio-ß-D-galactopyranoside and was bound to glutathione-Sepharose beads. Recombinant murine (r)MCP-1 was eluted from the Sepharose beads by the treatment with PreScission protease. The obtained rMCP-1 showed a single band on SDS-PAGE with a molecular mass of 19.6 kDa (Fig. 1 ). The molecular mass was closely identical to that of rMCP-1, whose chemotactic activity was confirmed (provided by Dr. Naofumi Mukaida, Kanazawa University, Ishikawa, Japan). After dialysis against PBS, the obtained rMCP-1 was used to immunize rabbits. pAb against MCP-1 was purified by extraction with n-caprylic acid from serum. The purity was determined with SDS-PAGE, and its titer was with ELISA. The specificity of anti-MCP-1 pAb was assessed by using MCP-1-, MIP-1{alpha}-, MIP-1ß-, and MIP-2-containing samples, which were obtained at 3 h after the third injection of apoptotic P388 cells. Before and after preincubation of the samples with anti-MCP-1 pAb at 37°C for 1 h, the protein levels of MCP-1, MIP-1{alpha}, MIP-1ß, and MIP-2 were measured with specific ELISAs. As the results show, only the level of MCP-1 was diminished after preincubation, confirming the specificity of anti-MCP-1 pAb.


Figure 1
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  		Figure 1. Purity of our rMCP-1. The GST-MCP-1 fusion protein bound to glutathione-Sepharose beads was cleaved with PreScission protease into GST (bound) and MCP-1 (soluble). The purity of MCP-1 was examined with 15% SDS-PAGE.

 
Neutralization of MCP-1
To neutralize MCP-1 activity, 2 mg anti-MCP-1 pAb was administered i.p. just before injection of apoptotic P388 cells. The amount of the pAb was considered to be sufficient, as 1.5 mg pAb suppressed leukocyte infiltration to the same extent (data not shown). Anti-GST pAb, which was prepared similarly as described above, was injected as a control.

Administration of rMCP-1 in neutrophil-depleted mice
To determine whether the CTL response in neutrophil-depleted mice is restored by rMCP-1, 0.5 µg or 1 µg rMCP-1 (provided by Dr. Naofumi Mukaida) was used [21 , 22 ]. They were administered 2 h after the last immunization.

Statistical analysis
Differences between experimental groups were analyzed by means of one-way factorial ANOVA (one-factor ANOVA) and the post-hoc test (Scheffe’s F) using Statcel (OMS Publishing, Saitama, Japan). When P < 0.05, the difference was considered statistically significant.

RESULTS

A time-dependent change of the number of leukocytes in the peritoneal cavity after immunization with apoptotic P388 cells
P388 cells were treated with etoposide to induce apoptosis as described in Materials and Methods. Apoptotic P388 cells (2x107 cells) were then injected into the peritoneal cavity of allogeneic C57BL/6 mice (H-2b) three times every 4 days, and we examined a time-dependent change of the number of leukocytes, including Gr-1+ cells, CD11b+ cells, and CD8+ cells, in the peritoneal cavity after the last immunization. As illustrated in Figure 2A (upper left), the leukocytes in PEC were classified into several populations based on Gr-1 and CD11b expression patterns. Neutrophils were identified as Gr-1high, CD11bint cells, whereas monocytes and macrophages were identified as Gr-1int, CD11bint cells and Gr-1-/int, CD11bhigh cells, respectively [23 , 24 ]. FSC versus SSC profiles of all cells, monocytes, and neutrophils were also shown in Figure 2A (upper right, lower left, and lower right, respectively). Neutrophils exhibited higher SSC and slightly lower FSC than monocytes. When neutrophils and monocytes were sorted with a cell sorter (EPICS Altra), Giemsa staining of each population indicated characteristic morphologies of neutrophil and monocyte (data not shown). Cells in the outside of the frames in Figure 2A (upper left) included eosinophils, B cells, mast cells, and apoptotic P388 cells, as evidenced by Giemsa, Congo Red, and antibody staining (data not shown).


Figure 2
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  		Figure 2. A time-dependent change of the number of leukocytes in the peritoneal cavity after the last immunization with apoptotic P388 cells. C57BL/6 mice were immunized by i.p. injection of 2 x 107 cells of apoptotic P388 cells three times every 4 days, and then PEC were recovered at indicated times (from 1 h to 5 days). (A) Characteristics of various leukocytes. To determine various cell types, PEC were recovered 12 h after the last immunization and analyzed flow cytometrically. The leukocytes were identified as neutrophil, monocyte, and macrophage with their expression of Gr-1 and CD11b, and each leukocyte was delineated with a framed rectangle (upper left). Forward-scatter (FSC) versus side-scatter (SSC) profiles of all cells (upper right), monocytes (lower left), and neutrophils (lower right) were shown. (B) A time-dependent change of the number of leukocytes in the peritoneal cavity after the last immunization. PEC were recovered at indicated times, and the number of each cell type was determined by flow cytometry. Data are expressed as means of five to seven mice.

 
We then examined a time-dependent change in the number of leukocytes in the peritoneal cavity after the last immunization with apoptotic cells (Fig. 2B) . The number of neutrophils reached a peak at 5 h and returned to a basal level after 1 day. The number of monocytes increased later than that of neutrophils and reached a peak at 12 h. The number of macrophages increased much later than those of neutrophils and monocytes and remained high even after 5 days. The number of CD8+ T cells increased later than that of macrophages and remained moderate even after 5 days.

Induction of CTL response by repeated injection of apoptotic P388 cells
Five days after each immunization with apoptotic cells, the cytotoxic activity in PEC was assessed against P388 cells (H-2d), A20 cells (H-2d), or EL4 cells (H-2b) by a standard 51Cr release assay. Although primary immunization did not induce significant cytotoxic activity (data not shown), strong cytotoxic activity against P388 cells was obtained after the third immunization (Fig. 3A ). Conversely, when necrotic cells instead of apoptotic cells were used in each immunization or in the last immunization, the cytotoxic activities were much weaker than that obtained by apoptotic cells (Fig. 3B) . The cytotoxic activity appears to be alloantigen-specific, as A20 cells but not EL4 cells were also lysed to the same extent as P388 cells (Fig. 3A) . The alloantigen-specific cytotoxic activity was also detected in splenocytes to a much lesser extent. The percent of specific release was only 35%, even at an effector:target cell ratio of 300:1 (Fig. 3C) . The cytotoxic activity in PEC was ascribed to CD8+ T cells, as anti-CD8 mAb and complement depleted virtually all the cytotoxic activity as compared with anti-CD4 mAb or control mAb (Fig. 3D) .


Figure 3
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  		Figure 3. Induction of alloantigen-specific CTL response by repeated injection of apoptotic P388 cells. (A) C57BL/6 mice (H-2b) were immunized by apoptotic P388 cells (H-2d) as described in Figure 2 . Five days after the last immunization, the cytotoxic activity in PEC was assessed against P388 cells (H-2d, •), A20 cells (H-2d, {diamond}), or EL4 cells (H-2b, {triangleup}). (B) C57BL/6 mice (H-2b) were immunized with apoptotic or necrotic P388 cells three times (apo cells x3, •, vs. nec cells x3, {triangleup}). Alternatively, mice were immunized with apoptotic P388 cells twice and then necrotic P388 cells (last nec cells, {diamond}). The cytotoxic activities in PEC were then assayed. (C) The cytotoxic activities in splenocytes were assayed against P388 cells (H-2d, •) and EL4 cells (H-2b, {triangleup}). (D) PEC taken from mice 5 days after the last immunization were treated with anti-CD8 mAb, anti-CD4 mAb, or control mAb and complement, and the remaining cytotoxic activity was examined at the effector:target cell ratio of 100:1. Each determination was carried out in triplicate. Data are expressed as means ± SE of four mice.

 
Relationship between neutrophil infiltration and CTL response
Our previous studies showed that injection of apoptotic P388 cells into the peritoneal cavity of CDF1 mice led to transient infiltration of neutrophils and that the infiltration of neutrophils was dependent on MIP-2 [18 , 19 ].

To determine whether neutrophils play a role in the CTL induction, we then examined the effect of neutrophil depletion by anti-Gr-1 mAb and that of inhibition of neutrophil infiltration by anti-MIP-2 pAb. As shown in Figure 4A , administration of anti-Gr-1 mAb but not control mAb (anti-HLA mAb) 1 day before injection of apoptotic P388 cells abrogated neutrophil infiltration completely, and administration of anti-MIP-2 pAb but not control antibody (anti-GST pAb) just after injection of apoptotic P388 cells inhibited infiltration of neutrophils significantly. These results were in good agreement with our previous results [18 , 19 ]. We then examined the effects of these treatments on the CTL induction. Administration of anti-Gr-1 mAb 1 day before injection of apoptotic P388 cells significantly inhibited the CTL activity as compared with that of control mAb (Fig. 4B) . Likewise, administration of anti-MIP-2 pAb reduced the CTL activity to a half-level as compared with control pAb (Fig. 4C) . When mice received anti-Gr-1 mAb 1 day after the last immunization, conversely, the CTL activity was not decreased as compared with mice receiving control mAb (Fig. 4D) . It should be noted that the number of neutrophils returned to a basal level 1 day after the injection of apoptotic cells (Fig. 2B) . These results indicated a close relation between CTL induction and neutrophil infiltration.


Figure 4
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  		Figure 4. Effect of suppression of neutrophil infiltraton on CD8+ T cell accumulation and CTL activity. (A) Suppression of neutrophil infiltration by anti-Gr-1 mAb or anti-MIP-2 pAb treatment. Anti-Gr-1 mAb (200 µg) or anti-HLA antibody as a control was administered i.p. 1 day before the last injection of apoptotic P388 cells. Anti-MIP-2 pAb (800 µg) or anti-GST antibody as a control was administered i.p. just before the last injection of apoptotic P388 cells. PEC were collected at 5 h, and the number of neutrophils was determined by flow cytometry. The results are expressed as means ± SE of four mice. *, P < 0.001. (B–D) Inhibition of CTL activity in the peritoneal cavity by anti-Gr-1 mAb or anti-MIP-2 pAb treatment. Mice received antibodies as above [anti-Gr-1 mAb, 1 day before (B) or after (D) the last immunization; anti-MIP-2 pAb (C), just before the last immunization]. Five days later, the CTL activity in PEC was assayed against P388 cells. Each determination was carried out in triplicate. (E) Inhibition of CD8+ T cell accumulation into the peritoneal cavity by anti-Gr-1 mAb or anti-MIP-2 pAb treatment. Mice were treated with antibodies as described above. Five days later, the number of CD8+ T cells in PEC was determined by flow cytometry. The results are expressed as means ± SE of five mice. As a control, mice were immunized without treatment with antibodies. *, P < 0.01; **, P < 0.05, respectively, as compared with controls. Data are expressed as means ± SE of four mice.

 
Furthermore, we examined the effects of administration of anti-Gr-1 mAb or anti-MIP-2 pAb on the number of CD8+ cells infiltrated into the peritoneal cavity. As shown in Figure 4E , the number of CD8+ cells was decreased significantly by administration of anti-Gr-1 mAb or anti-MIP-2 pAb as compared with that of control pAb. In contrast, administration of anti-Gr-1 mAb 1 day after the last immunization did not cause a decrease in the number of CD8+ cells as compared with that of control mAb. Accordingly, these results suggested that neutrophils infiltrating at the early period (within 1 day) are required for accumulation of CD8+ T cells into the peritoneal cavity at a later period.

There are reports that administration of anti-Gr-1 mAb reduced the number of memory-type CD8+ T cells (CD8+, CD44high, CD62Lhigh) [25 , 26 ]. However, it is unlikely that memory-type CD8+ T cells are affected by administration of anti-Gr-1 mAb in this study, as the administration 1 day after the last immunization did not result in a decrease in the CTL activity and the number of CD8+ T cells (Fig. 4D and 4E) .

Production of chemokines in the peritoneal cavity
We then hypothesized that neutrophils may be involved in the production of chemokines for attracting CD8+ T cells. As MIP-1{alpha}, MIP-1ß, and MCP-1 are secreted by neutrophils and are capable of attracting CD8+ T cells, we determined the levels of these chemokines in the peritoneal lavage fluid at various times after the last immunization. As shown in Figure 5 , MIP-1{alpha}, MIP-1ß, and MCP-1 were detected as early as 1 h after immunization and reached peaks at 2–3 h (824.5±213.5 pg/head, 4503.4±192.3 pg/head, and 10,264.7±2565.8 pg/head, respectively). They returned to basal levels after 1 day. The time kinetics was quite similar with that of neutrophils, but none of them was detected when the number of CD8+ T cells started to increase (after 1 day). We then examined whether the infiltrating neutrophils are involved in the production of MIP-1{alpha}, MIP-1ß, and MCP-1 in the peritoneal cavity. As shown in Figure 6 , the production of these chemokines was decreased significantly by administration of anti-Gr-1 mAb 1 day before the last immunization or that of anti-MIP-2 pAb just before the last immunization as compared with that of control pAb. Among them, MCP-1 production was suppressed most significantly by these treatments.


Figure 5
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  		Figure 5. Kinetics of chemokine production in the peritoneal cavity after the last immunization. The peritoneal lavage fluid was collected at indicated times (from 1 h to 5 days) after the last immunization, followed by determination of the levels of MIP-1{alpha}, MIP-1ß, and MCP-1 by specific ELISAs. The results are expressed as means ± SE of four mice.

 

Figure 6
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  		Figure 6. Effect of suppression of neutrophil infiltration on chemokine production. Mice were treated with anti-Gr-1 mAb or anti-MIP-2 pAb as described in Figure 4 . The levels of chemokines in peritoneal lavage fluid were determined 5 h after the last immunization. The results are expressed as means ± SE of four mice. *, P < 0.001; **, P < 0.005; and ***, P < 0.05, respectively, as compared with controls.

 
Effect of anti-MCP-1 pAb on infiltration of CD8+ T cells into the peritoneal cavity and the CTL induction
We then examined the role of MCP-1 in the infiltration of CD8+ T cells into the peritoneal cavity after the last immunization. To neutralize MCP-1 activity, 2 mg anti-MCP-1 pAb was administered i.p. just before the last immunization. We then determined the numbers of leukocytes in the peritoneal cavity, namely neutrophils, monocytes, macrophages, and CD8+ T cells. As shown in Figure 7A , neutralization of MCP-1 resulted in significant decreases in the numbers of monocytes, macrophages, and CD8+ T cells in the peritoneal cavity. The number of neutrophils was not decreased significantly by anti-MCP-1 pAb. Administration of anti-MCP-1 pAb also inhibited the CTL activity in PEC significantly, as compared with that of control pAb (Fig. 7B , top). In addition, the CTL activity in splenocytes was inhibited by administration of anti-MCP-1 pAb (Fig. 7B , bottom). These results indicate that MCP-1 plays a role in infiltration of CD8+ T cells as well as the CTL induction.


Figure 7
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  		Figure 7. Inhibitory effects of anti-MCP-1 pAb on a time-dependent change of the number of leukocytes and CTL induction. (A) Effect of anti-MCP-1 pAb on a time-dependent change of the number of various leukocyte populations in the peritoneal cavity. Anti-MCP-1 pAb (2 mg) or anti-GST pAb as a control was administered i.p. just before the last immunization. PEC were collected at indicated times (from 5 h to 5 days), and the number of each cell population was determined by flow cytometry. The results are expressed as means ± SE of three to four mice. *, P < 0.001; **, P < 0.005; and ***, P < 0.05, respectively, as compared with controls. (B) Effect of anti-MCP-1 pAb on CTL induction. Mice were treated in the same way as described above. Five days after the last immunization, the CTL activity in PEC or splenocytes was assayed against P388 cells. Each determination was carried out in triplicate. Data are expressed as means ± SE of four mice.

 
Effect of murine MCP-1 on infiltration of CD8+ T cells and CTL induction in neutrophil-depleted mice
We then examined whether infiltration of CD8+ T cells and CTL induction in neutrophil-depleted mice were restored by the i.p. administration of rMCP-1. As shown in Figure 8A and 8B , the number of macrophages decreased with anti-Gr-1 mAb after 1 day or 5 days, whereas they were restored significantly by administration of rMCP-1. In the same way, rMCP-1 also restored the number of CD8+ T cells significantly (Fig. 8C and 8D) . Administration of rMCP-1 (1 µg) also restored the CTL activities in PEC (Fig. 8E , left) and splenocytes (Fig. 8E , right) to similar levels as those obtained by control pAb. These results further support that MCP-1 links neutrophils with infiltration of CD8+ T cells as well as the CTL induction.


Figure 8
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  		Figure 8. Effects of rMCP-1 on the number of macrophages and CD8+ T cells and CTL induction in neutrophil-depleted mice. (A–D) Mice were treated with anti-Gr-1 mAb or anti-HLA antibody (Control Ab) 1 day before the last immunization, as described in Figure 4 . Then, 0.5 µg or 1 µg rMCP-1 was administered i.p. at 2 h after the last immunization. PEC were collected at 1 day (A, C) or 5 days (B, D) after the last immunization, and the numbers of macrophages (A, B) and CD8+ T cells (C, D) were determined by flow cytometry. The results are expressed as means ± SE of three to seven mice. *, P < 0.01; **, P < 0.05, as compared with neutrophil-depleted mice (anti-Gr-1 mAb). (E) rMCP-1 (1 µg) was administered into neutrophil-depleted mice in the same way as described above. Five days after the last immunization, the CTL activity in PEC or splenocytes was assayed against P388 cells. Anti-Gr-1 mAb + rMCP-1, {blacksquare}; anti-Gr-1 mAb, •; control antibody, {circ}; no antibody, {diamond}. Each determination was carried out in triplicate. Data are expressed as means ± SE of four mice.

 
DISCUSSION

Recently, much attention has been paid to the role of neutrophils as a regulator of the immune response against bacterial infection and tumor [3 4 5 6 ]. For example, in gastric Helicobacter infection, neutrophil depletion caused a delay of the bacterial clearance from the stomach and markedly decreased Th1 immune response to Helicobacter [3 ]. Likewise, in Leishmania major infection, neutrophil depletion resulted in exacerbation of pathology in susceptible BALB/c mice as compared with resistant C3H/HeJ mice, and this was associated with changes in the profiles of Th1 cytokines and Th1 chemoattractive chemokine expression in draining lymph node cells and/or macrophages [5 ]. Conversely, in some models of antitumor immunity, neutrophils appear to play a role as effector cells [27 , 28 ]. However, to our knowledge, it is not known whether infiltrating neutrophils are involved in CTL induction upon immunization with apoptotic tumor cells. We therefore examined it in this study by using an allogeneic CTL response as a model.

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity [18 , 19 ]. The infiltration of neutrophils was mainly caused by MIP-2, which was produced by macrophages ingesting apoptotic P388 cells [18 ]. In this study, we confirmed our previous results and extended them by showing that neutrophils, monocytes/macrophages, and CD8+ T cells appeared sequentially in the peritoneal cavity after injection of late apoptotic P388 cells. Furthermore, we demonstrated that infiltration of CD8+ T cells and CTL induction by immunization are greatly dependent on infiltration of neutrophils.

In this model, immunization with necrotic cells did not induce a potent CTL response. Although dendritic cells (DC) have been shown to be able to process apoptotic and necrotic tumor cells for antigen presentation and to prime CD8+ and CD4+ T cells, recent studies have indicated that the antigenic difference between apoptotic and necrotic tumor cells clearly influences the ability of DC to present antigen to T cells [29 30 31 32 ]. Some researchers showed that apoptotic tumor cells induced a stronger CTL response than necrotic cells [31 , 32 ], which appears to be consistent with our results.

Among chemokines, MCP-1, MIP-1{alpha}, and MIP-1ß are involved in the induction of an antigen-specific CTL response into the local sites of inflammation [10 11 12 13 ]. MCP-1 binds to CCR2 to accumulate monocytes/macrophages [33 , 34 ], DC [35 ], T cells [8 , 9 ], and NK cells [34 ], thereby playing an important role in innate and adaptive immunity. MIP-1{alpha} and -ß accumulate T cells [8 , 11 , 36 ], monocytes/macrophages [14 , 16 ], DC [35 ], neutrophils [37 ], and NK cells [16 ]. We demonstrated in this study that MCP-1 production was largely dependent on neutrophil infiltration as compared with MIP-1{alpha} and -ß production. Moreover, administration of anti-MCP-1 pAb caused suppression of increases of monocytes, macrophages, and CD8+ T cells in the peritoneal cavity. Anti-MCP-1 pAb also caused significant inhibition of CTL activity. Furthermore, administration of rMCP-1 was able to restore the infiltration of CD8+ T cells and CTL induction in neutrophil-depleted mice. Taken together, these findings suggest that MCP-1 appears to link infiltration of neutrophils with CTL induction.

Recent studies indicated a role of MCP-1 as a regulator of T cell responses [9 , 13 ]. For example, in Cryptococcus neoformans infection, MCP-1-neutralizing antibody significantly reduced macrophage and T cell (CD8+ and CD4+ T cell) recruitment into the lungs and diminished antigen-specific IFN-{gamma} production significantly [9 ]. In another study, MCP-1 secreted by tumor cells was shown to attract melanoma patients’ CTLs toward apoptotic tumor cells in vitro [38 ].

It is not known at present whether MCP-1 attracts CD8+ CTLs directly in this study. Leukotrience B4 (LTB4), which is known as an early neutrophil chemoattractant [39 ], has been shown to accumulate effector CD8+ T cells, which express BLT1, the high-affinity receptor for LTB4, at sites of inflammation [40 41 42 ]. Moreover, MCP-1 was reported to enhance the production of LTB4 from peritoneal macrophages of normal mice in a dose-dependent manner [43 ]. Hence, the possibility is that MCP-1 induces the production of LTB4 to accumulate effector CD8+ CTLs into the peritoneal cavity after immunization with apoptotic cells.

In conclusion, we demonstrated in this study that upon i.p. injection of allogeneic tumor cells at a late stage of apoptosis, infiltrating neutrophils cause accumulation of CD8+ CTLs into the peritoneal cavity, which is largely dependent on MCP-1. Thus, a better understanding of the mechanism of how neutrophils regulate CTL induction could have an important implication for adaptive immune responses.

Received June 14, 2006; revised September 6, 2006; accepted October 12, 2006.

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