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Originally published online as doi:10.1189/jlb.0807578 on December 21, 2007

Published online before print December 21, 2007
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(Journal of Leukocyte Biology. 2008;83:640-647.)
© 2008 by Society for Leukocyte Biology

Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines

Nicolas Kesteman*, Georgette Vansanten*, Bernard Pajak*, Sanna M. Goyert{dagger} and Muriel Moser*,1

* Université Libre de Bruxelles, Institut de Biologie et Médecine Moléculaires, Gosselies, Belgium; and
{dagger} North Shore University Hospital/New York University School of Medicine, Manhasset, New York, USA

1Correspondence: Laboratoire de Physiologie Animale, Université Libre de Bruxelles, Rue des Prof. Jeener et Brachet, 12, 6041 Gosselies, Belgium. E-mail: mmoser{at}ulb.ac.be


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ABSTRACT
 
There is increasing evidence that neutrophils are involved in the regulation of adaptive immunity. We therefore tested whether these cells may colocalize with T lymphocytes in lymphoid organs. Our results demonstrate that administration of the microbial product LPS induces the migration of neutrophils in the spleen from the red pulp and the marginal zone to the area of the white pulp where T cells reside. This movement is CD14-dependent, whereas the recruitment of neutrophils in the peritoneal cavity is increased in the absence of CD14. Our data further suggest the involvement of the chemokine MIP-2 and keratinocyte-derived chemokine and their receptor CXCR2. We conclude that neutrophils may interact with naïve T cells upon infection/inflammation and that the migration of neutrophils in the lymphoid organs and in the periphery is regulated differently by a signal transduced by CD14

Key Words: cell trafficking • KC • MIP-2 • LIX • dendritic cells


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INTRODUCTION
 
Immune reactivity usually occurs in lymphoid organs, which provide anatomical structures guiding cellular interaction and receptor–ligand engagement. In particular, the splenic white pulp is structurally similar to a lymph node and contains discrete areas of T and B cells, whereas the red pulp comprises mainly macrophages and granulocytes [1 ]. We and others [2 , 3 ] have shown previously that most dendritic cells (DC) are distributed in the two splenic compartments in the steady state and migrate massively to the T cell area upon systemic administration of LPS. This movement parallels a functional maturation process and results in the colocalization of T lymphocytes and APC, a sequence of events that is considered as an important, early step of the immune response.

There is increasing evidence that in addition to DC, other cell populations of the innate immune system shape adaptive immunity. In particular, neutrophils, which are key components of the inflammatory response, recruit APC and regulate their function, transport antigen, and control T cell expansion and differentiation [4 5 6 7 8 9 ]. Several reports have underscored an efficient cooperation between neutrophils and T lymphocytes. Neutrophils can synergize with T lymphocytes in protecting young, susceptible rats from fatal experimental malaria [10 ]. Neutrophil depletion modulates the Th1/Th2 dichotomy in several infection models. In experimental Chagas’ disease, their depletion in BALB/c resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines [11 ]. In contrast, in C57BL/6, neutrophil depletion induced resistance to the disease and enhanced the expression of Th1 cytokines [11 ]. Likewise, a role for neutrophils in driving Th1-type responses has been illustrated in a mouse model of Legionella pneumophila pneumonia [12 ], as well as in Candida-specific immunity [7 , 13 ]. Although neutrophils seem to favor mainly Th1 responses, one report showed that neutrophils played an early role in the induction of the nonprotective Th2 response, which develops in Balb/c mice following infection with Leishmania major, and their depletion led to partial resolution of the footpad lesions induced by L. major [14 ]. Furthermore, i.p. injection of allogeneic tumor cells at a late stage of apoptosis induced accumulation of CD8+ CTL into the peritoneal cavity, and this immune response was linked to the infiltration of neutrophils [15 ]. Finally, several reports have demonstrated that early neutrophil influx in organ transplants favored T cell-mediated rejection (for review, see refs. [16 17 18 19 20 ]).

As the instructive role of neutrophils in adaptive immunity is likely to require physical interaction with lymphocytes, an interesting question is where this contact may occur. Access of the white pulp has been shown to be restricted to lymphocytes and DC, and the distribution of neutrophils was almost exclusively confined to the red pulp [1 ]. We therefore examined whether neutrophils may undergo a dynamic process as described for DC and migrate from the red pulp to the white pulp, where naïve T cells reside. We found that administration of LPS induced the migration of neutrophils to the T cell area as early as 90 min after injection before the movement of DC.


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MATERIALS AND METHODS
 
Mice and in vivo treatment
BALB/c and C57BL/6 mice (6–8 weeks old) were from Harlan (Horst, Nederland). C57BL/6 and BALB/c CD14–/– mice were generated as described [21 ]. Dr. Shizuo Akira (Osaka University, Japan) kindly provided the C57BL/6 MyD88 strain. CXCR2 knockout (KO) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All mice were housed in our pathogen-free facility, and the experiments were performed in compliance with the relevant laws and institutional guidelines.

Mice were injected i.v. or i.p. with 1, 5, or 10 µg Ultra Pure Escherichia coli LPS (strain 0111:B4, InvivoGen, San Diego, CA, USA) in 200 µl pyrogen-free PBS or with 25 µg anti-CD3 mAb (hamster mAb 145-2C11 to mouse CD3{epsilon} [22 ], purified in our laboratory). Control animals were injected with the same volume of PBS. To transiently deplete neutrophils in vivo, some mice were injected i.p. with 15 µg RB6-8C5 mAb (produced in-house).

Flow cytometry and cell sorting
Single-cell suspensions from spleens were incubated for 20 min at 4°C with saturating doses of 2.4G2 (a rat anti-mouse FcR mAb, American Type Culture Collection, Manassas, VA, USA) and specific antibodies in staining buffer (0.5% BSA in PBS/NaN3). Data were collected on multicolor flow cytometry (FACSCanto II, Becton Dickinson, Mountain View, CA, USA). The following antibodies were used for staining: FITC-conjugated 1A8 (anti-Ly6G, Pharmingen, San Diego, CA, USA) and PE-conjugated anti-CXCR2 (clone 242216, R&D Systems, Minneapolis, MN, USA). Cells were gated according to size and scatter to eliminate dead cells and debris from analysis. For sorting, splenic CD11b+ cells were first enriched by positive selection using magnetic MACS separation columns (Miltenyi Biotec, Bergish-Gladbach, Germany), further labeled with anti-Ly6G-PE (clone 1A8) and anti-F4.80-FITC (clone BM8, Pharmingen), and sorted with a FACSVantage (Becton Dickinson).

RT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, Frederick, MD, USA) and was primed with oligo(dT) for first-strand cDNA synthesis (Moloney murine leukemia virus RT, Invitrogen), according to the manufacturer’s instructions. The quantitative PCR core kit for SYBR Green (Eurogentec, Belgium) and GeneAmp 5700 sequence detection system (PE Applied Biosystems, Foster City, CA, USA) were used for real-time PCR. The following primer pairs were used: RPL32, forward 5'-GGCACCAGTCAGACCGATAT-3' and reverse 5'-CAGGATCTGGCCCTTGAAC-3'; CXCL-2, forward 5'-CTGTCCCTCACCGGAAGAAC-3' and reverse 5'-CGAGGCACATCAGGTACGAT-3'; CXCL-5, forward 5'-CCGCTGGCATTTCTGTTG-3' and reverse 5'-CGTTGCGGCTATGACTGA-3'; and CXCL-1, forward 5'-TGTCAGTTATTTATTGAAAGTCGTCTT-3' and reverse 5'-CGAGACGAGACCAGGAGAAA-3'. Transcript amounts were normalized to those of the gene encoding ribosomal protein 32, a housekeeping gene.

Immunohistochemistry
Frozen processing
Spleens were harvested and frozen at –80°C in CryoBlock (Klinipath, Nederland; see Figs. 1 2 and 4 ). Frozen sections (8 µm-thick) were fixed in methanol at 20°C for 7 min and transferred to PBS.


Figure 1
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  		Figure 1. LPS administration induces the migration of 1A8+ neutrophils to the T cell area in the white pulp. BALB/c mice were injected i.v. with PBS (A) or with 5 µg LPS (B–E). At the time indicated, the spleens were harvested and cryosections double-stained with anti-Ly6G (blue) and anti-CD90 mAb (red). The original magnification was x1000. These results are representative of four independent experiments. (A) The inset (right panel) illustrates the polymorphonuclear morphology of sorted 1A8+ cells (Giemsa staining).


Figure 2
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  		Figure 2. CD14 is critical for LPS-induced migration of neutrophils. WT (A, C, and E) and CD14 KO BALB/c mice mice (B, D, and F) were injected i.v. with PBS (A and B), 5 µg LPS (C and D), or 25 µg anti-CD3 (aCD3) mAb (E and F). Spleens were harvested after 90 min and cryosections double-stained with anti-Ly6G (blue) and anti-CD90 mAb (red). Three experiments were performed with similar results.


Figure 4
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  		Figure 4. MyD88 is critical for LPS-induced migration of neutrophils. WT (A and C) and MyD88 KO (B and D) C57BL/6 mice were injected i.v. with 5 µg LPS. Ninety minutes later, the spleens were harvested and cryosections double-stained with anti-Ly6G (blue) and anti-CD90 mAb (red). These results are representative of four independent experiments.

Wax processing
Spleens were fixed in Immunohistofix (Aphase, Belgium), embedded in Immunohistowax (Aphase), and sectioned at 3–6 µm, as described previously (see Fig. 7 ) [23 ].


Figure 7
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  		Figure 7. LPS-induced migrations of neutrophils and DC are independent events. (A) C57BL/6 mice were depleted or not (Control) of neutrophils by i.p. injection of 15 µg RB6-8C5 and injected i.v. 1 day later with PBS or 10 µg LPS. Spleens were harvested 6 h later, and wax-processed sections were stained with fluorescein-conjugated 1A8 (upper panels) or with biotin-conjugated N418 revealed by alkaline phosphatase (lower panels). Three experiments were performed with similar results. (B) WT or MyD88 KO mice were injected with PBS or 5 µg LPS. Spleens were harvested 6 h later, and sections were stained for CD11c in red and for CD86 in blue. Three experiments were performed with similar results.

The sections were treated for 30 min at room temperature with blocking reagent (Boehringer, Germany; 1% in PBS) to saturate the sites of nonspecific reactions. The slides were washed in PBS, treated with H2O2, 3% in PBS for 30 min at room temperature to block endogenous peroxydase, washed, and stained with biotinylated anti-Thy (anti-CD90, clone T24.1, produced in-house), biotinyled anti-CD11c mAb (clone N418, produced in-house), and/or FITC-conjugated rat anti-mouse Ly-6G mAb (clone 1A8, Pharmingen) for 2–3 h at room temperature. The slides were washed further in PBS and incubated with avidin-biotin-peroxidase complex or avidin-biotin-alkaline phosphatase complex (both from Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) and/or anti-fluorescein-alkaline phosphatase (Roche, Germany) for 30 min at room temperature and washed in PBS. The alkaline phosphatase activity was revealed in blue using substrate kit III from Vector Laboratories. The peroxidase activity was revealed using amino-3-ethyl-9-carbazole (AEC; Sigma Chemical Co., St. Louis, MO, USA) giving red precipitates.

For double-staining CD11c-CD86, CD86 was first stained in blue using biotinylated anti-CD86 (clone GL1, eBioscience, San Diego, CA, USA), avidin-biotin-alkaline phosphatase complex, and substrate kit III (Vector Laboratories). The biotin in excess was blocked with the blocking kit (Vector Laboratories), and CD11c was stained in red using biotinyled anti-CD11c mAb, avidin-biotin-peroxidase complex, and AEC.

Slides were mounted in Aquatex (Merck, Germany), and digitized images were captured with Leica DM 4000B (Germany) and analyzed with Adobe Photoshop (San Jose, CA, USA).


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RESULTS
 
LPS induces the rapid migration of neutrophils from the marginal zone to the T cell area of the spleen
LPS was administered to mice, and neutrophils and T cells were visualized in cryosections from spleen 15–90 min later using specific mAb (Fig. 1 ). In PBS-injected mice, the majority of neutrophils [stained in blue with 1A8 mAb, see Fig. 1A , inset (right panel)] was detected in the marginal zone between the red and the white pulp, as well as in the red pulp (Fig. 1A) . Of note, the injection of LPS led to a redistribution of 1A8+ cells, which were detected in association with T lymphocytes (stained in red with T24.1 mAb) 90 min after LPS injection (Fig. 1E) . Using the program Image J [National Institutes of Health (NIH), Bethesda, MD, USA], we calculated that an average of 16% of neutrophils (1A8+) migrated to the T cell area after LPS administration, as compared with 1.6% in PBS-injected mice (Table 1 ). The analysis of neutrophils at intermediate time-points (Fig. 1B 1C 1D) strongly suggests that neutrophils have migrated from the marginal zone to the area where T cells reside.


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  		Table 1. Proportion of Neutrophils in the White Pulp

CD14 is critical for the migration of neutrophils in the spleen but not in the peritoneal cavity
Among the receptors for LPS identified, CD14 is a glycoprotein, expressed on monocytes, DC, and neutrophils as a GPI-anchored molecule. It is associated with TLR4, a signal-transducing receptor for LPS.

We evaluated the role of CD14 in vivo using genetically deficient mice [21 ]. LPS was administered to wild-type (WT) or CD14 KO mice, and spleen sections were examined for 1A8 and Thy1.2 expression. The data (Fig. 2D ) clearly show that neutrophils remained in the marginal zone in LPS-injected CD14 KO mice, indicating that CD14 is critical for their migration. The absence of movement was not a result of an intrinsic defect in their migratory capacity, as neutrophils migrated after anti-CD3 mAb injection (Fig. 2F) , as shown previously for DC [24 ]. By contrast, CD14-deficient mice had much higher numbers of neutrophils in the peritoneal cavity than control mice early after LPS injection (Fig. 3A ), an observation consistent with a previous study [25 ]. TLR4 has been shown to use two downstream signaling pathways, one depending on the adaptor MyD88 and one signaling through the adaptor Toll-IL-1R domain-containing adaptor-inducing IFN-β. We therefore tested the role of MyD88 and similarly found that migration of neutrophils was enhanced in the peritoneal cavity (Fig. 3B) but strongly impaired in the spleen (Fig. 4 ) of mice deficient for this adaptor molecule.


Figure 3
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  		Figure 3. Increased numbers and infiltration of neutrophils in the peritoneal cavity of CD14 KO and MyD88 KO mice. WT (A and B), CD14 KO (A), or MyD88 KO (B) C57BL/6 mice were injected i.p. with 5 µg LPS. Three hours later, the peritoneal cavity was washed with cold PBS, and cells were stained with FITC-conjugated anti-Ly6G mAb. Data are shown as mean ± SD of at least two mice tested individually. The numbers of neutrophils harvested in each group are indicated above the bars. These data are from one experiment representative of three independent experiments.

The chemokine receptor CXCR2 and its ligand(s) are involved in LPS-induced migration
Neutrophil migration is regulated by signaling mechanisms activated by chemokine receptors to direct cell migration along the concentration gradient. In particular, the chemokines MIP-2 (also named CXCL-2), keratinocyte-derived chemokine (KC; CXCL-1), and LPS-induced CXC chemokine (LIX; CXCL-5) have been shown to bind to the cell surface receptor CXCR2. We analyzed the expression of CXCR2 on neutrophils from spleen cells of mice injected with PBS or LPS using double staining for 1A8 and CXCR2 and found that CXCR2 expression was restricted to 1A8+ cells (not shown). This expression rapidly decreased after LPS injection (Fig. 5 ), an observation in line with previous studies showing that desensitization of chemokine receptors of the G-protein-coupled receptors regulates agonist stimulation. The analysis of neutrophils in CXCR2 KO mice after LPS injection showed that 50% of the white pulps were invaded with neutrophils after LPS injection, as compared with 90% in WT animals (Supplemental Fig. 1). Although the results were difficult to interpret as a result of a major increase in neutrophil numbers, they suggest that this receptor has a redundant role in the migration of neutrophils. We next measured the mRNA coding for the chemokines MIP-2, KC, and LIX in the spleen of LPS-injected mice. Our results (Fig. 6A ) show that mRNA encoding KC and MIP-2 was detected as early as 10 min after treatment, peaked at 45 min, and decreased thereafter. The LIX mRNA increased more progressively and peaked 60 min after LPS injection. In all experiments, the increase in MIP-2 and KC mRNA ranged from 300- to 900-fold, whereas the LIX mRNA expression increased by approximately tenfold. Much lower levels of mRNA coding for MIP-2, KC, and LIX were detected in CD14 KO mice (Fig. 6B) . To identify the cell populations secreting these chemokines in vivo, we purified (by FACSsorting) 1A8+ and F4/80+ cells from the spleen of LPS-injected mice. Our results in Figure 6C indicate that MIP-2 and KC are produced by distinct cell populations: MIP-2 was secreted mainly by neutrophils, whereas KC was produced by F4/80+ cells, which include mainly monocytes and macrophages (Fig. 6C) .


Figure 5
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  		Figure 5. CXCR-2 is down-regulated following LPS injection. WT C57BL/6 mice were injected i.v. with 10 µg LPS. The spleens were harvested at the time indicated, and spleen cells were double-stained with FITC-conjugated 1A8 and PE-conjugated anti-CXCR2 mAb. Negative controls include cells stained with FITC-conjugated 1A8 mAb only (Unstained). Data are shown as mean fluorescence intensity (MFI) of CXCR2 expression on gated 1A8+ cells. These data are representative of three independent experiments.


Figure 6
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  		Figure 6. MIP-2 and KC are expressed by distinct cell populations. WT (A–C) and CD14 KO (B) BALB/c mice were injected i.v. with PBS or 10 µg LPS (A), 1 µg LPS (B), and 5 µg LPS (C), and the spleen cells were harvested at the indicated time-point (A) or after 90 min (B) and 45 min (C). mRNA coding for chemokines KC, MIP-2, and LIX was quantified by PCR analysis in enriched populations of splenic neutrophils (94% Ly6G+) and monocytes (95% F4/80+). Data were normalized to those of RPL32 mRNA. Data are expressed as arbitrary units relative to the sample WT PBS arbitrarily set to one and are representative of four independent experiments.

DC and neutrophils migrate independently
We next tested whether neutrophils, which migrate early after LPS injection and produce chemoattractants for DC (see later discussion), direct the subsequent movement of DC to the white pulp. Our data in Figure 7A indicate that the migration of DC to the white pulp is similar in control mice (left panels) and in mice depleted of neutrophils by anti-Gr1 injection (right panels). Most neutrophils were depleted after RB6-8C5 injection, as illustrated in the upper panels (Fig. 7A) for LPS-injected mice. As we have shown that the adaptor MyD88 was critical for the migration of neutrophils (Fig. 4) , we analyzed the movement of DC after LPS inoculation in MyD88-competent or -deficient mice. Our results clearly show that MyD88 was not required for the movement of DC (Fig. 7B) . In addition, DC similarly up-regulated their expression of CD86, a hallmark of the phenomenon of DC maturation [2 ]. Collectively, these observations indicate that the LPS-induced migrations of neutrophils and DC in the T cell zone are two independent events.


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DISCUSSION
 
More direct evidence for a role of neutrophils in the induction and regulation of adaptive immunity would be provided by showing that neutrophils can localize in close proximity with lymphocytes, including naïve T lymphocytes, which are confined in lymphoïd organs. We therefore analyzed the spleen, the largest secondary lymphoïd organ, which consists of two compartments: the red pulp involved in the clearance of bacteria and dead blood cells from the bloodstream and the white pulp, densely populated with lymphocytes, which plays a major role in the development of immune responses.

The objective of this work was to test whether neutrophils can reach the white pulp in inflammatory conditions. Our results indicate that neutrophils, which are found mainly in the red pulp and at the margin between the red and white pulp, migrate to the white pulp in vivo in response to the bacterial product LPS. These observations extend a few studies [4 , 14 , 26 ], illustrating the migration of neutrophils to the lymph nodes in infected or immunized mice by showing that neutrophils may reach the splenic area where naïve T cells reside.

We show further that the LPS receptor CD14, which is associated with TLR4, and its adaptor MyD88 are required for the migration of neutrophils in the spleen. By contrast, the recruitment of neutrophils in the peritoneal cavity was much higher in the absence of CD14 (over a 50-fold increase) and to a lesser extent, of MyD88 (threefold increase). These results indicate that the migration of neutrophils in a lymphoïd organ and in a peripheral tissue is differentially regulated by a signal transduced by CD14. In agreement with this observation, evidence has been provided for an early LPS-induced neutrophil infiltration in CD14-deficient mice, which is delayed in control animals [25 ]. Similarly, CD14 KO mice exhibited enhanced infiltration of monocytes and neutrophils in various models [27 28 29 30 ]. Of note, CD11b was shown responsible for the CD14-independent pathway [29 ]. We have found that CD11b was up-regulated on splenic and peritoneal neutrophils early after LPS injection (not depicted) and may therefore be involved in the movement of neutrophils. Finally, the cytokine TNF, known to direct the migration of several cell populations, does not seem critical, as a similar movement to the white pulp was noticed in TNF-{alpha} KO mice (not depicted). Collectively, these observations underscore the unique role of CD14 and MyD88 in inducing the colocalization of neutrophils and T lymphocytes in the spleen and confirm that CD14 may trigger a negative-feedback loop, which interferes with the rapid neutrophil influx in the peritoneal cavity.

It is unclear whether LPS activates neutrophils directly or whether chemokines released by other sets of cells indirectly trigger the movement of neutrophils. Our data suggest that the receptor CXCR2 is activated following LPS administration, as it is down-regulated rapidly a mechanism of regulation well known for chemokine receptors of the G-protein-coupled receptor family. We have also found that MIP-2 and KC, two ligands for CXCR2, were produced within minutes after LPS injection. Of note, MIP-2 is released by neutrophils, whereas KC is produced by monocytes, suggesting direct and indirect triggering of neutrophils, although few monocytes were present in the white pulp (not depicted). It would be interesting to test the role of autologous chemotaxis, a mechanism recently described, whereby autocrine chemokine secretion directs tumor cells to chemotact in the direction of flow [31 ].

Our data using CXCR2 KO mice suggest that this receptor has a redundant role in the migration of neutrophils. Several reports have shown an increased number of neutrophils associated with impaired migration in the absence of CXCR2 and have described CXCR2 ligands as essential mediators of host defense [32 33 34 ]. The role of these chemokines is illustrated further by the finding that MIP-2 and KC were found significantly higher in brains of CD14–/– than of WT mice in a model of murine Streptococcus pneumoniae meningitis.

The precise role of neutrophils in adaptive immunity is still unclear. Several reports (see later discussion) have shown that neutrophils may induce the maturation and/or the migration of DC, a hypothesis that would be consistent with the colocalization of DC and neutrophils in the T cell area between 3 h and 6 h after LPS administration. Toxoplasma gondii-stimulated neutrophils released factors chemotactic for DC and triggered IL-12 and TNF-{alpha} production in vivo [35 ]. Neutrophils strongly clustered with immature DC and when activated, induced the maturation of DC. This interaction was driven by the binding of the DC-specific, C-type lectin DC-specific ICAM-grabbing nonintegrin to the β2-integrin membrane-activated complex 1 [36 ]. Interestingly, a recent report demonstrates that live neutrophils can deliver activating signals to immature DC, whereas via cell contact, live and apoptotic neutrophils can transfer heat-killed Candida albicans antigens to DC, allowing the latter to stimulate T lymphocytes [5 ]. We did not obtain any evidence suggesting that neutrophils may direct the migration of DC in our experimental setting. Indeed, transient neutrophil depletion did not affect LPS-driven migration of DC. The independent movement of neutrophils and DC was illustrated further by the analysis of MyD88 KO mice, in which LPS induced the migration of DC but not of neutrophils.

In addition to their capacity to stimulate DC, neutrophils themselves may produce cytokines, in particular, IL-12 [13 , 37 , 38 ]. Rapid recruitment of neutrophils containing prestored IL-12 was observed 4 h after in vivo infection with T. gondii [37 ]. The physiological relevance of IL-12 production was shown by the higher production of IL-12 by neutrophils from healer mice infected with a live vaccine strain of C. albicans, as compared with nonhealer mice infected with the virulent strain [13 ]. Neutrophils can also transport antigens and are endowed with antigen-presenting capacity in some circumstances [4 5 6 , 9 , 26 , 39 ]. In particular, Maletto et al. [26 ] have shown that when FITC-labeled OVA was injected into the footpad of OVA/CFA-immunized mice, the main OVA-FITC+ cells recruited in draining popliteal lymph nodes were neutrophils.

In conclusion, our data show that access to the splenic white pulp is not restricted to lymphocytes and DC and that neutrophils may colocalize with T lymphocytes. This movement is strictly dependent on CD14 and MyD88, in contrast to the early recruitment in the peritoneal cavity, which appears inhibited by a TLR4/CD14 transduction pathway. Our observations suggest that the LPS-induced migration to the white pulp may be triggered by MIP-2 produced by neutrophils themselves, a mechanism reminiscent of autologous chemotaxis [31 ], and/or by KC produced by monocytes or macrophages. The colocalization of neutrophils with DC and T lymphocytes would be consistent with a regulatory role in the early steps of the adaptive immune response and in particular, on the function of DC and the development of Th subsets.


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ACKNOWLEDGEMENTS
 
The Laboratory of Animal Physiology is supported by grants of the Fonds National de la Recherche Scientifique (FNRS)/Télévie, by the Fonds de la Recherche Fondamentale Collective, by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, European Grants (DC-VACC, DC-THERA, Cancerimmunotherapy), and by the Belgian Cancer Foundation. N. K. had fellowships from the Fonds pour la Formation à la Recherche dans l’Industrie et l’Agriculture; M. M. is research director from the FNRS. We thank M. de Heusch for experimental help and useful discussions, O. Leo for helpful suggestions, and P. Veirman for animal care.

Received August 28, 2007; revised November 9, 2007; accepted November 11, 2007.


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