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Highly Biased Type 1 Immune Responses in Mice Deficient in LFA-1 in Listeria monocytogenes Infection Are Caused by Elevated IL-12 Production by Granulocytes ¹

Masashi Emoto^2,3,*, Mamiko Miyamoto^2,*, Yoshiko Emoto^2,*, Izumi Yoshizawa^*, Volker Brinkmann, Nico van Rooijen and Stefan H. E. Kaufmann^*

^* Department of Immunology and Central Support Unit Microscopy, Max-Planck- Institute for Infection Biology, Berlin, Germany; and Department of Cell Biology and Immunology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN- producers in the liver and spleen of LFA-1^-/- mice were markedly increased compared with heterozygous littermates and V14⁺NKT cell-deficient mice, and NK cells were major IFN- producers. Numbers of IL-12 producers were also markedly elevated in LFA-1^-/- mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN- production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN--secreting NK cells in the liver of LFA-1^-/- mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1^-/- mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1^-/- mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN- secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leukocyte function-associated Ag-1 is an / hetero-dimeric transmembrane glycoprotein consisting of an L subunit (CD11a) and a 2 subunit (CD18) expressed on the surface of virtually all leukocytes, albeit at varying levels (1). In the mouse the major counter-receptors for LFA-1 are ICAM-1 (CD54) and ICAM-2 (CD102), which are differentially expressed on various cells, such as leukocytes, epithelial cells, endothelial cells, and fibroblasts (1). LFA-1 is considered important in a broad range of cellular events, ranging from firm adhesion of leukocytes to endothelial cells initiating leukocyte transmigration and extravasation (1) to functional activation of various inflammatory cells, including NK cells (2, 3, 4, 5, 6, 7, 8) and T cells (6, 9, 10, 11, 12, 13, 14).

Listeria monocytogenes belongs to the family of facultative intracellular bacteria, which are able to propagate in professional phagocytes (15, 16). Immediately after systemic infection, the vast majority of microorganisms are entrapped in the liver, ingested, and destroyed by professional phagocytes (15, 16, 17). Granulocytes play a central role at this early stage, and the majority of bacteria are eliminated by this cell population (15, 18, 19, 20, 21). Effective protection against L. monocytogenes is contingent on activation of type 1 immune responses (15, 16). IFN- and IL-12 are key cytokines that induce type 1 immune responses; hence, these cytokines are essential for protection against L. monocytogenes infection (15, 16). Since NK1⁺ cells and macrophages, including Kupffer cells, have been suggested to be responsible for prompt IFN- or IL-12 production, respectively, these cells are considered important for the early host defense against L. monocytogenes (15, 16, 21, 22). Infiltration of these cells into sites of inflammation is an essential prerequisite for elimination of bacteria. As a corollary, cell adhesion molecules, notably LFA-1 should participate in host defense against intracellular microbial pathogens.

NK1⁺ cells segregate into two major populations, namely NKT cells and NK cells. NKT cells represent a specialized T cell subset that expresses NK cell markers, such as NKR-P1 (NK1.1), in addition to T cell markers, including CD3 (23). The majority of NKT cells express an invariant TCR-chain encoded by V14 rearranged to J281, and their development depends on ₂-microglobulin-associated CD1d (23). V14⁺NKT cells are abundant in the liver (24, 25), where they promptly secrete large amounts of IFN- as well as IL-4 after TCR stimulation (24, 26, 27). In contrast, NK cells develop independently of ₂-microglobulin, lack surface expression of CD3, and produce IFN-, but not IL-4 (28), although these cells are also abundant in the liver (29). Because accumulating evidence suggests that V14⁺NKT cells are essential for NK cell activation (30, 31) and because LFA-1 is essential for accumulation of the majority of V14⁺NKT cells in the liver (32), LFA-1 could critically influence the induction of type 1 immune responses in the liver.

Experiments using specific mAb to block LFA-1 in vivo suggest that signaling through LFA-1 plays an important role in polarization toward type 1 immune responses (33, 34, 35, 36, 37). Yet it remains elusive whether LFA-1 is indeed involved in type 1 polarization in vivo. In the present study we assessed the influence of LFA-1 on polarization toward type 1 immune responses during listeriosis using LFA-1^-/- mice. We found that LFA-1^-/- mice develop elevated type 1 immune responses and demonstrate that elevated type 1 immune responses in these mice are caused by IL-12 secreted by granulocytes at the early stage of listeriosis. We assume that granulocytes are not only central to rapid bacterial clearance, but also regulate the ensuing acquired immune response of type 1. On the molecular level, LFA-1 profoundly regulates the protective immune response against infection with a bacterial pathogen by controlling granulocyte infiltration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Breeding pairs of LFA-1^-/- mice (6) and J281^-/- mice (38) were provided by Drs. R. Schmits (University of Saarland, Homburg, Germany) and M. Taniguchi (RIKEN, Yokohama, Japan), respectively. The mutants backcrossed onto C57BL/6 (LFA-1^-/- and LFA-1^+/-, fourth generation; J281^-/-, eighth generation) were maintained under specific pathogen-free conditions at our animal facilities, and weight- and generation-matched male mice were used at 8–10 wk of age. Backcross onto a given mouse strain can influence characteristic phenotypic features of gene knockout mutants (39). Hence, LFA-1^+/-, rather than wild-type, mice were used as controls for LFA-1^-/- mice to ensure adequate comparability of genetic backgrounds.

Antibodies

mAbs against CD3 (145-2C11), FcR (2.4G2), IFN- (R4-6A2 and XMG1.2), Ly6G (RB6-8C5), IL-12 (p40/p70; C17.8), and IL-12 (p40; C15.6.7) were purified from hybridoma culture supernatants. Anti-IFN- mAb (XMG1.2) and anti-IL-12 (p40) mAb (C15.6.7) were biotinylated, and anti-CD3 mAb and anti-Ly6G mAb were conjugated with FITC by conventional methods. Biotinylated anti-NK1.1 mAb (PK136), PE-conjugated anti-Mac-1 mAb (M1/70), FITC-conjugated anti-rat IgG2b (R35-38), PE-conjugated anti-Ly6G mAb (RB6-8C5), PE-conjugated rat IgG1 (R3-34), and PE-conjugated anti-IFN- mAb (XMG1.2) were purchased from BD PharMingen (Hamburg, Germany). F4/80 mAb (CI:A3-1) and Cy2-conjugated anti-rat IgG Ab were obtained from Serotec (Oxford, U.K.) and Jackson ImmunoResearch Laboratories (West Grove, PA), respectively.

Bacteria and infection

L. monocytogenes (strain EGD) recovered from infected liver were grown in tryptic soy broth (Difco, Detroit, MI) at 37°C for 18 h, and aliquots were frozen at -80°C until use. The final concentration of viable bacteria was enumerated by plate counts with tryptic soy agar (Difco). Mice were infected i.v. with 1.5 x 10⁴ L. monocytogenes. Heat-killed L. monocytogenes (HKL) ⁴ were prepared by heating bacteria in a water bath at 80°C for 3 h. HKL were washed three times with PBS and frozen at -20°C until used.

Cell preparation and flow cytometry

Hepatic leukocytes (HL) were prepared as described previously (24). Splenocytes were prepared by conventional methods. After blocking with anti-FcR mAb, cells were stained with conjugated mAbs at 4°C for 15 min, and biotinylated mAbs were visualized with streptavidin (SA)-conjugated CyChrome (BD PharMingen). After staining, cells were washed with PBS containing 0.1% BSA (Serva, Heidelberg, Germany) and 0.1% sodium azide (Merck, Darmstadt, Germany), and then fixed with 1% paraformaldehyde (Merck). Stained cells were acquired by FACScan (BD Biosciences, Mountain View, CA) and analyzed with CellQuest software.

ELISPOT assay

The frequencies of IFN-- and IL-12-producing cells were measured by ELISPOT methods as described previously (26, 40) with slight modifications. Briefly, appropriate dilutions of cells were cultured overnight in the presence or the absence of HKL in ELISPOT plates (Millipore, Eschborn, Germany) coated with anti-IFN- mAb (R4-6A2) or anti-IL-12 mAb (C17.8). Plates were then washed and incubated with biotinylated anti-IFN- mAb (XMG1.2) or biotinylated anti-IL-12 mAb (C15.6.7), respectively, at 37°C for 2 h. For developing spots, SA-conjugated alkaline phosphatase (Dianova, Hamburg, Germany) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets (Sigma-Aldrich, Deisenhofen, Germany) were used. The numbers of cytokine-secreting cells were estimated by counting spots using a dissecting microscope.

ELISA

Serum levels of IL-12 (p40) were determined by ELISA as described previously (41). In brief, serum samples were incubated in immunoassay plates (Nunc, Copenhagen, Denmark) precoated with anti-IL-12 (p40/p70) mAb (C17.8). After washing, plates were incubated with biotinylated anti-IL-12 (p40) mAb (C15.6.7), followed by SA-conjugated alkaline phosphatase (Dianova) and the chromogen p-nitrophenyl phosphate (Sigma-Aldrich). The cytokine concentration in each sample was determined using serially diluted mouse rIL-12 (Genzyme, Alzenau, Germany).

In vivo treatment

For neutralization of IL-12, mice were treated i.p. with 500 µg of anti-IL-12 mAb (C17.8) 2 h before infection. Multilamellar liposome-encapsulated dichloromethylene bisphosphonate was prepared as described previously (42). Cl2MBP was a gift from Roche (Mannheim, Germany). To deplete tissue macrophages, mice were injected i.v. with 200 µl of liposome-encapsulated dichloromethylene bisphosphonate (containing 1 mg of Cl2MBP) suspended in PBS 2 days before infection as described previously (42, 43, 44). As a control, mice were injected i.v. with 200 µl of liposome-encapsulated PBS. To deplete granulocytes, mice were treated i.p. with 150 µg of anti-Ly6G mAb 1 day before infection as described previously (45, 46). Depletion of almost all (>98%) Kupffer cells and granulocytes in the liver was verified by immunohistochemistry and/or flow cytometry using F4/80 mAb and anti-Ly6G mAb, respectively (data not shown). Labeling with FITC-conjugated anti-rat IgG2b (for flow cytometry) or Cy2-conjugated anti-rat IgG (for immunohistochemistry) did not stain cells from anti-Ly6G mAb-treated mice, excluding residual coating of the cells with mAb (data not shown). Isotype-matched mAb purified by the same procedure as that used for specific mAb or PBS used for mAb purification were used as a control, and it was verified that the outcome of mAb treatment was not caused by LPS contamination in mAb or PBS.

Intracellular cytokine staining

Intracellular IFN- staining was performed as described previously (47) with slight modifications. In brief, cells were incubated for 3 h in the presence of GolgiPlug (5 µg/ml; BD PharMingen). Cells were then stained with mAb against cell surface markers. After fixation and permeabilization with Cytofix/Cytoperm (BD PharMingen), intracellular IFN- staining was conducted using PE-conjugated anti-IFN- mAb. As a control, PE-conjugated rat IgG1 was used. Stained cells were acquired by FACScan and analyzed with CellQuest software.

Immunohistochemistry

Liver specimens were embedded in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands), frozen, and cut on a Leica Cryotome (Leica Microsystems, Bensheim, Germany). Sections (3–5 µm) were air-dried, fixed with acetone, rehydrated, and treated with blocking buffer (PBS containing 1% BSA and 0.05% Tween 20) for 15 min. Sections were then incubated with F4/80 mAb diluted in blocking buffer at a concentration of 10 µg/ml at 37°C for 30 min. After washing with PBS, the sections were incubated with Cy2-conjugated anti-rat IgG Ab.

Cell sorting

Ly6G⁺ cells were positively sorted by a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. In brief, HL were stained with PE-conjugated anti-Ly6G mAb for 15 min at 4°C after blocking and subsequently incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min at 6°C. Cell suspensions were applied to a MS+/RS+ column (Miltenyi Biotec), and then Ly6G⁺ cells were enriched by flushing out. The purity of Ly6G⁺ cells was always >90%.

Statistical analysis

Statistical significance was determined by post hoc multiple range test. A value of p < 0.05 was regarded as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Markedly elevated IFN- production by NK cells during L. monocytogenes infection in the absence of LFA-1

To examine whether LFA-1 participates in IFN- production during L. monocytogenes infection, we compared the frequencies of IFN--producing cells in the liver and spleen of LFA-1^-/- and LFA-1^+/- mice by ELISPOT assay. The numbers of IFN--producing cells increased among HL and splenocytes from both LFA-1^-/- and LFA-1^+/- mice on day 1 postinfection (pi) and further increased after in vitro stimulation with HKL (Fig. 1). The numbers of IFN- producers were significantly higher in LFA-1^-/- mice than in LFA-1^+/- mice regardless of the presence of HKL. Note that the frequencies of IFN--producing cells were higher among HL than splenocytes. Thus, at the early stage of listeriosis IFN--producing cells were more efficiently induced in the absence of LFA-1.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Frequencies of IFN--producing cells in livers and spleens of LFA-1-/- and LFA-1+/-mice before and after L. monocytogenes infection. Mice were infected i.v. with 1.5 x 104 L. monocytogenes, and HL and splenocytes were prepared on days 0 and 1 pi. Cells were incubated in the presence or the absence of HKL in ELISPOT plates precoated with anti-IFN- mAb. Data are expressed as the mean of duplicate cultures (SD, <10%). *, p < 0.01; **, p < 0.001 (LFA-1-/- vs LFA-1+/-). Determinations were performed four times with comparable results. SFC, spot-forming cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Proportions of IFN--producing cells among CD3+NK1.1+ and CD3-NK1.1+ cells in livers of LFA-1-/-, LFA-1+/-, and J281-/- mice after L. monocytogenes infection and the influence of endogenous IL-12 neutralization or granulocyte depletion. LFA-1-/-, LFA-1+/-, and/or J281-/- mice were left untreated or were treated i.p. with anti-IL-12 mAb (500 µg) or anti-Ly6G mAb (150 µg). Two hours (for anti-IL-12 mAb-treated group) or 1 day (for anti-Ly6G mAb-treated group) later, mice were infected i.v. with 1.5 x 104 L. monocytogenes, and HL were prepared on day 1 pi. Cells were incubated in the presence of GolgiPlug for 3 h and stained with FITC-conjugated anti-CD3 mAb and biotinylated anti-NK1.1 mAb, followed by SA-conjugated CyChrome. Cells were further stained with PE-conjugated anti-IFN- mAb. Data are displayed as histograms after gating on the indicated cell populations. The numbers in the histograms represent the percentages of IFN--producing cells. IFN--producing cells were virtually undetectable in all mouse strains examined before infection. Rat IgG1 mAb (isotype-matched mAb for anti-IFN- mAb) staining was always <0.5%, thus excluding nonspecific staining. No significant difference was found among rat IgG2a (isotype-matched mAb for anti-IL-12 mAb)-treated group, rat IgG2b (isotype-matched mAb for anti-Ly6G mAb)-treated group, and untreated group in another experiment (data not shown). Determinations were performed at least twice with comparable results.

IFN- production by NK cells in LFA-1^-/- mice is prevented by endogenous IL-12 neutralization

IL-12 plays a central role in NK cell activation and induction of IFN--producing cells (50, 51, 52, 53). We therefore examined the influence of endogenous IL-12 neutralization on IFN- secretion from liver NK cells by intracellular IFN- staining. The frequencies of IFN--producing cells among liver CD3^-NK1⁺ cells from LFA-1^-/- mice were markedly diminished by anti-IL-12 mAb treatment (p < 0.01; Fig. 2). We conclude that IL-12 promotes L. monocytogenes-induced IFN- production by NK cells in the absence of LFA-1.

Elevated frequencies of IL-12 producers in LFA-1^-/- mice following L. monocytogenes infection

We compared the frequencies of IL-12 producers in the liver and spleen as well as serum levels of IL-12 by ELISPOT assay and ELISA, respectively, in LFA-1^-/- and LFA-1^+/- mice. The numbers of IL-12-producing cells increased among HL and splenocytes from both LFA-1^-/- and LFA-1^+/- mice on day 1 pi and were further increased by in vitro stimulation with HKL (Table II). The numbers of IL-12 producers were significantly higher in LFA-1^-/- mice than in LFA-1^+/- mice regardless of HKL stimulation. Note that the frequencies of IL-12-producing cells were higher among HL than among splenocytes. In contrast, serum levels of IL-12 were comparable in LFA-1^-/- and LFA-1^+/- mice on day 1 pi, although they were low (mean serum levels: LFA-1^-/-, 7.6 ng/ml; LFA-1^+/-, 7.7 ng/ml; data not shown). Thus, following L. monocytogenes infection, IL-12 producers were more efficiently induced in inflamed sites at the early stage of listeriosis in the absence of LFA-1.

We attempted to determine the cellular source of IL-12 in LFA-1^-/- mice following L. monocytogenes infection. Because macrophages have been considered to be a major source of IL-12 in listeriosis (50), we assessed the influence of tissue macrophage depletion on IL-12 production. The numbers of IL-12 producers were slightly, although not significantly, increased in LFA-1^-/- and LFA-1^+/- mice by tissue macrophage depletion (Table II). These results argue against a major role of tissue macrophages (e.g., Kupffer cells) in IL-12 production during listeriosis regardless of LFA-1 expression.

We have recently found that infiltration of granulocytes into the liver is markedly higher in LFA-1^-/- mice than in LFA-1^+/- mice following L. monocytogenes infection (45). We raised the question of whether granulocytes are a major source of IL-12 in LFA-1^-/- mice. The numbers of IL-12 producers among HL were markedly diminished in both LFA-1^-/- and LFA-1^+/- mice by granulocyte depletion with the anti-Ly6G mAb (Table II). Note that numerical reduction of IL-12 producers by anti-Ly6G mAb treatment was more profound in LFA-1^-/- mice than in LFA-1^+/- mice. In parallel to the numerical reduction of IL-12-producing cells, IFN- production by NK cells was also diminished by Ly6G⁺ cell depletion (p < 0.01; Fig. 2). These results suggest that Ly6G⁺ cells, presumably granulocytes, regulate IFN- production by NK cells via IL-12 production.

To directly verify whether granulocytes infiltrating the liver at the early stage of listeriosis secrete IL-12, Ly6G⁺ cells were positively sorted from the liver of L. monocytogenes-infected LFA-1^-/- and LFA-1^+/- mice, and the frequencies of IL-12 producers were determined by ELISPOT assay. Substantial numbers of IL-12-producing cells were detected among Ly6G⁺ cells purified from LFA-1^-/- mice, which were further increased by in vitro stimulation with HKL (Fig. 3). Considerable numbers of IL-12 producers were also detected among Ly6G⁺ cells purified from LFA-1^+/- mice, although the numbers were significantly lower than in LFA-1^-/- mice. These results point to granulocytes as the major source of IL-12 in the liver at the early stage of listeriosis and suggest a more dominant role for granulocytes as IL-12 producers in LFA-1^-/- mice compared with LFA-1^+/- mice.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Frequencies of IL-12-producing cells among liver Ly6G+ cells purified from LFA-1-/- and LFA-1+/- mice after L. monocytogenes infection. Mice were infected i.v. with 1.5 x 104 L. monocytogenes, and HL were prepared on day 1 pi. Cells were stained with PE-conjugated anti-Ly6G mAb, and Ly6G+ cells were positively sorted. Cells were incubated in the presence or the absence of HKL in ELISPOT plates precoated with anti-IL-12 mAb. Data are expressed as the mean of duplicate cultures (SD, <10%). *, p < 0.01; **, p < 0.001 (LFA-1-/- vs LFA-1+/-). Determinations were performed twice with comparable results. SFC, spot-forming cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It could be speculated that the elevated type 1 immune responses in the liver of LFA-1^-/- mice during listeriosis were due to reduced numbers of V14⁺NKT cells in the liver. However, the numbers of IFN--producing cells in the liver of V14⁺NKT cell-deficient mice were not elevated following L. monocytogenes infection. We therefore consider it unlikely that the highly biased type 1 immune response observed in LFA-1^-/- mice was due to a numerical reduction of V14⁺NKT cells in the liver. We assume that V14⁺NKT cells are dispensable for NK cell activation, at least in listeriosis. Yet, we do not preclude contribution of V14⁺NKT cells to NK cell activation under physiologic or certain pathologic inflammatory conditions. We have recently reported that the numbers of V14^-NKT cells, which are potent IFN- producers, are also diminished in the liver of LFA-1^-/- mice (44). We therefore do not formally exclude the possibility that NK cells compensate for the reduction of the NKT cell population in LFA-1^-/- mice.

Evidence has been presented that LFA-1 participates in functional activation of NK cells (2, 3, 4, 5, 6, 7, 8). In contrast, the numbers of IFN--producing NK cells were markedly elevated in LFA-1^-/- mice following L. monocytogenes infection. It is likely that LFA-1 is dispensable for activation of NK cells. Yet, 1) the frequencies of IL-12-producing cells were markedly higher in LFA-1^-/- mice than in LFA-1^+/- mice following L. monocytogenes infection, 2) IL-12 plays a central role in IFN- production from NK cells (50, 51, 52, 53), and 3) endogenous IL-12 neutralization markedly diminished IFN- production by NK cells in the liver of LFA-1^-/- mice following L. monocytogenes infection. Hence, we consider it likely that LFA-1 contributes to polarization toward type 1 immune responses, which is, however, covered by the more profound effects of the type 1-promoting cytokine IL-12. We consider it unlikely that the higher numbers of IFN--producing NK cells in LFA-1^-/- mice are caused by differential activation of NK cells in these mice, because Mac-1 expression on NK cells was comparable in homozygotes and heterozygotes. Moreover, expression levels of IL-12R- and IL-12-chains on NK cells are comparable in LFA-1^-/- and LFA-1^+/- mice (8), suggesting that higher numbers of IFN--producing NK cells in the absence of LFA-1 are probably not due to increased IL-12 responsiveness. Although serum levels of IL-12 were comparably low in LFA-1^-/- and LFA-1^+/- mice on day 1 pi, differences increased at later time points (M. Emoto, M. Miyamoto, and Y. Emoto, unpublished observation). We therefore assume that local IL-12 production influences systemic cytokine responses at later time points.

LFA-1 has been shown to play a major role in adhesion of NK cells to vascular endothelium (4) and in transendothelial migration of NK cells (5). In contrast to these findings, numbers of NK cells infiltrating the liver following L. monocytogenes infection were higher in LFA-1^-/- mice than in LFA-1^+/- mice. This discrepancy could be due to different experimental systems (i.e., in vitro vs in vivo). Inflammation is caused by different cells and cytokines that contribute to inflammation in a hierarchic order. Hence, NK cells are differentially influenced by the microenvironment. Although we do not formally exclude the participation of LFA-1 in transmigration and extravasation of NK cells under physiologic or certain pathologic conditions, we assume that LFA-1 is redundant for infiltration of NK cells into inflamed sites.

Myeloid-derived APCs, such as monocytes, macrophages, and dendritic cells, are potent IL-12 producers (53), and macrophages are considered a major source of IL-12 in listeriosis (50, 51). In contrast, the frequencies of IL-12 producers were slightly increased by depletion of tissue macrophages. We cannot provide an answer for the increased frequencies of IL-12 producers after macrophage depletion. Nevertheless, our results argue against a pivotal role for tissue macrophages in IL-12 production during listeriosis. Because granulocyte depletion resulted in the reduction of IL-12 producers, and because high frequencies of IL-12 producers were detected among granulocytes, we assume that these cells play a crucial role not only as phagocytes, but also as regulators of the ensuing course of infection by producing the type 1-inducing cytokine, IL-12. Indeed, granulocyte-derived IL-12 has been suggested to play a role in polarizing immunity toward type 1 immune responses in Candida and Toxoplasma infection (54, 55, 56, 57). Because IFN- secretion by NK cells was not completely prevented by granulocyte depletion, we consider it likely that cells other than granulocytes also participate in IFN- production by NK cells. Indeed, serum levels of IL-12 following infection were significantly (p < 0.05) higher in granulocyte-depleted LFA-1^-/- mice than in nondepleted LFA-1^+/- mice (M. Emoto, M. Miyamoto, and Y. Emoto, unpublished observation).

We have recently shown that LFA-1^-/- mice are far more resistant to L. monocytogenes infection and that increased resistance to L. monocytogenes in the absence of LFA-1 is caused by neutrophilia, followed by accelerated liver infiltration of granulocytes (45). Since serum levels of G-CSF as well as IL-17 were markedly increased in the absence of LFA-1 (45), we assume that highly biased type 1 immune responses in the absence of LFA-1 are caused by neutrophilia, facilitating increased granulocyte infiltration into the liver promptly after L. monocytogenes infection.

In conclusion, we describe an inhibitory role for LFA-1 in polarization toward type 1 immune responses. Whereas the role of granulocytes as major effectors of the early host response to L. monocytogenes is well established, our data reveal that they also serve as a major regulatory cell in polarization toward type 1 immune responses by producing IL-12.

	Acknowledgments

 
We thank Drs. Rudolf Schmits and Masaru Taniguchi for breeding pairs of LFA-1^-/- and J281^-/- mice, Daniela Groine-Triebkorn for screening of mice, and Beatrix Fauler and Ulrike Reichard for help with histological procedures.

	Footnotes

 
¹ This work was supported by a grant from the German Science Foundation (SFB421).

² M.E., M.M., and Y.E. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Masashi Emoto, Department of Immunology, Max-Planck-Institute for Infection Biology, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail address: emoto{at}mpiib-berlin.mpg.de

⁴ Abbreviations used in this paper: HKL, heat-killed L. monocytogenes; HL, hepatic leukocytes; pi, postinfection; SA, streptavidin.

Received for publication April 7, 2003. Accepted for publication August 11, 2003.

	References

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