The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 6846 -6853
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jia, T.
Right arrow Articles by Pamer, E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jia, T.
Right arrow Articles by Pamer, E. G.

Additive Roles for MCP-1 and MCP-3 in CCR2-Mediated Recruitment of Inflammatory Monocytes during Listeria monocytogenes Infection1

Ting Jia*,{dagger}, Natalya V. Serbina*, Katharina Brandl*, Maggie X. Zhong*, Ingrid M. Leiner*, Israel F. Charo{ddagger} and Eric G. Pamer2,*,{dagger}

* Infectious Diseases Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; {dagger} Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10065; and {ddagger} Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA 94158


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokine receptor-mediated recruitment of inflammatory cells is essential for innate immune defense against microbial infection. Recruitment of Ly6Chigh inflammatory monocytes from bone marrow to sites of microbial infection is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. Although CCR2–/– mice are markedly more susceptible to Listeria monocytogenes infection than are wild-type mice, MCP-1–/– mice have an intermediate phenotype, suggesting that other CCR2 ligands contribute to antimicrobial defense. Herein, we show that L. monocytogenes infection rapidly induces MCP-3 in tissue culture macrophages and in serum, spleen, liver, and kidney following in vivo infection. Only cytosol invasive L. monocytogenes induce MCP-3, suggesting that cytosolic innate immune detection mechanisms trigger chemokine production. MCP-3–/– mice clear bacteria less effectively from the spleen than do wild-type mice, a defect that correlates with diminished inflammatory monocyte recruitment. MCP-3–/– mice have significantly fewer Ly6Chigh monocytes in the spleen and bloodstream, and increased monocyte numbers in bone marrow. MCP-3–/– mice, like MCP-1–/– mice, have fewer TNF- and inducible NO synthase-producing dendritic cells (Tip-DCs) in the spleen following L. monocytogenes infection. Our data demonstrate that MCP-3 and MCP-1 provide parallel contributions to CCR2-mediated inflammatory monocyte recruitment and that both chemokines are required for optimal innate immune defense against L. monocytogenes infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokines play a central role in homeostatic trafficking and positioning of immune cells and also provide guidance and direction to immune cells responding to inflammatory challenges (1). By binding to chemokine receptors and triggering intracellular signaling pathways that induce cell movement (2), chemokines orchestrate many aspects of innate and adaptive immune responses. For example, CCR2 and its chemokine ligand MCP-1 play pivotal roles in monocyte recruitment during infection or under other inflammatory conditions (3, 4, 5, 6, 7, 8, 9, 10, 11), and CX3CR1 and its ligand CX3CL1 (fractalkine) facilitate monocyte and macrophage trafficking in atherogenesis (12, 13). CCR2, CCR5, CXCR3, and CX3CR1 contribute to rapid NK cell mobilization that occurs in inflammatory conditions (14, 15, 16, 17, 18). CCR7 and its ligands, CCL19 and CCL21, direct the trafficking of T cells, B cells (19, 20), and activated dendritic cells (DCs)3 (21) to lymph nodes, a process that promotes priming and activation of naive T cells (22, 23) and colocalization of B and T cells within lymph nodes (24, 25). CCR5 and its ligands facilitate efficient CD8 T cell priming by promoting migration of naive CD8 T cells toward mature, Ag-bearing dendritic cells (26).

Cellular trafficking is a complex process. Directionality is provided by chemokine gradients that are established within tissues by interactions between chemokines and glycosaminoglycans (GAGs) (27, 28). Although GAG association is not required for in vitro chemokine activity, mutations within human MCP-1 that impair GAG binding inhibit MCP-1-mediated monocyte recruitment in vivo (29). Because chemokines differ in their affinity and specificity for tissue GAGs, it has been postulated that GAGs, by selectively binding distinct chemokines, play an essential role in directing leukocyte trafficking (30). Association with GAGs can also promote chemokine oligomerization, which also promotes chemokine-mediated leukocyte recruitment (28, 31). In addition to GAG association and oligomerization, shear forces have also emerged as a factor contributing to chemokine activity (32). In a low-flow, shear-free environment, surface-bound CCL21 promoted sustained T cell motility without activating integrins. However, when shear forces were applied, integrins were activated by CCL21 and resulted in arrest of T cells. Thus, this mechanism allows chemokines to trigger cell extravasation at specific sites in the vasculature, under high-flow conditions, while enhancing the mobility of trafficking cells in static compartments.

Chemokine and chemokine receptors play important roles during bacterial infection. Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that causes severe disease in immunocompromised hosts (33). L. monocytogenes virulence depends upon the pore-forming protein listeriolysin O (LLO), which enables bacterial escape from the phagosome and entry into the host cell cytoplasm (34). Early immune defense against L. monocytogenes infection requires CCR-2-mediated recruitment of inflammatory monocytes from the bone marrow (11) and differentiation of recruited monocytes into TNF- and inducible NO synthase (iNOS)-producing DCs (Tip-DCs) at sites of infection (35). In mice lacking CCR2 or MCP-1, Tip-DC recruitment is markedly diminished in spleen, resulting in enhanced in vivo bacterial growth that overwhelms the host (10).

Although the importance of CCR2 in immune defense against L. monocytogenes infection is well established, less is known about the relative contributions of CCR2 ligands to innate immune defense. In addition to MCP-1, three other CC-chemokines, MCP-2, MCP-3, and MCP-5, can bind CCR2 and trigger signaling. MCP-3 was identified as a protein with monocyte chemotactic activity that copurified with MCP-1 from stimulated human osteosarcoma cells (36). MCP-3 is structurally and functionally similar to MCP-1 and is a potent in vitro chemoattractant for monocytes (36), T cells (37), and NK cells (38). Recently, an MCP-3–/– mouse strain was generated and demonstrated that MCP-3 serves as an important CCR2 ligand that enhances in vivo monocyte recruitment (39).

In the present study, we characterize the expression of MCP-3 during L. monocytogenes infection, and investigate the relative contributions of MCP-3 and MCP-1 to monocyte recruitment and innate immune defense. We demonstrate that MCP-3 is rapidly induced following L. monocytogenes infection and that MCP-3–/– mice, like MCP-1–/– mice, are more susceptible to in vivo infection. Increased susceptibility of MCP-3–/– mice correlates with decreased inflammatory monocyte recruitment from bone marrow into circulation. Our data demonstrate, for the first time, that MCP-3 plays a critical role in immune defense against L. monocytogenes infection by mediating inflammatory monocyte recruitment.


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

All mice used in this study were bred at Memorial Sloan-Kettering Research Animal Resources Center. Generation of MCP-1–/–, MCP-3–/–, and CCR2–/– mice was previously described (4, 39, 40). All mice were backcrossed at least 10 generations onto the C57BL/6 background. Mice were infected i.v. with 3000 L. monocytogenes strain 10403S, 5 x 105 attenuated ActA-deficient L. monocytogenes strain DP-L1942, 108 attenuated LLO-deficient L. monocytogenes strain DP-L2161, or 109 heat-killed L. monocytogenes (HKL) prepared as decribed previously (41). At indicated times following infection, spleens were harvested and dissociated in PBS containing 0.05% Triton X-100, and bacterial CFUs were determined by plating on brain-heart infusion agar plates.

Cell culture

Macrophages were grown from bone marrow precursors in antibiotic-free DMEM medium supplemented with 30% supernatant from L cell fibroblasts and 20% FBS. On day 5, cells were harvested in ice-cold PBS, plated at 2–4 x 105/well in 96-well flat-bottom plates, and various bacterial strains or 108/ml HKL were added to the cells. Bacteria were grown to log phase (A600 of 0.1), pelleted at 10,000 rpm for 10 min, and resuspended in PBS before addition to cells. Wild-type and LLO-deficient bacteria were added to the cells at 5:1 bacteria-cell ratio. Total volume in the wells during infection was 200 µl in 96-well plates. Infection was allowed to proceed for 30 min, at which time extracellular bacteria were washed away, and gentamicin-containing media was added to each well to prevent extracellular bacterial growth. Supernatants were collected 2, 4, and 6 h postinfection to assay for chemokines.

Flow cytometry

At various times after infection, spleens were collected and dissociated and were digested with 0.3% collagenase type 4 (Worthington). Bone marrow cells were collected from mouse femurs. The following Abs were purchased from BD Pharmingen: anti-CD11b-PerCP (M1/70), anti-Ly6C-FITC (AL-21), and anti-TNF-FITC (MP6-XT22). Goat anti-iNOS Ab (M-19) was purchased from Santa Cruz Biotechnology, and FITC-anti-goat IgG was purchased from Jackson ImmunoResearch Laboratories. For FACS analysis, a large forward light scatter/side light scatter gate was drawn to include lymphocyte/monocyte populations. Intracellular staining for iNOS was performed by staining cells for cell surface markers, fixing in 2% paraformaldehyde, permeabilizing with Perm/Wash Buffer (BD Pharmingen), and incubating with goat anti-iNOS Ab followed by FITC-anti-goat IgG. For intracellular TNF staining, splenocytes were stimulated in vitro with 108/ml HKL in the presence of brefeldin A for 4 h and processed according to the manufacturer’s protocol (Cytofix/Cytoperm, Pharmingen).

ELISA

Murine MCP-1 was quantified using an ELISA kit from BD Biosciences, and murine MCP-3 were quantified using an ELISA kit from Bender MedSystems. To obtain lysates for chemokine assays, organs were harvested at indicated times following infection, macerated in ice-cold PBS containing 0.01% Triton X-100, and centrifuged at 10,000 x g.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MCP-3 is rapidly induced following L. monocytogenes infection

Our previous studies demonstrated that MCP-1 expression contributes to CCR2-mediated monocyte recruitment (10) during L. monocytogenes infection, and mice lacking CCR2 or MCP-1 are highly susceptible to L. monocytogenes infection (3, 10). Although the expression of MCP-1 following L. monocytogenes infection has been characterized, very little is known about the expression of MCP-3. Therefore, as a first step, we investigated the in vivo induction of MCP-1 and MCP-3 following L. monocytogenes infection. As shown in Fig. 1, we found that the kinetics of MCP-1 and MCP-3 induction are similar, with dramatic induction of both chemokines in spleen, serum, and liver 24 and 48 h following infection. We noticed that there was a higher level of MCP-1 than MCP-3 in the spleen 48 h postinfection. Induction of MCP-3 in spleen, serum, liver, and kidney was maximal 48 h following infection. Although the kinetics of MCP-1 and MCP-3 induction share many similarities, some significant differences were also detected. For one, while MCP-1 levels decrease between 48 and 72 h of infection, MCP-3 levels in spleen and liver remain elevated. Additionally, levels of MCP-3 in the kidney are quite high while levels of MCP-1 in the kidney remain relatively low throughout infection. Whether this disparity reflects differences in expression or trapping of these chemokines in the kidney requires further investigation.


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 1. Kinetics of in vivo MCP-1 and MCP-3 expression following L. monocytogenes infection. C57BL/6 mice were infected i.v. with 3000 live L. monocytogenes. At the indicated times, spleens, livers, and kidneys were removed and serum was harvested and MCP-1 and MCP-3 levels were determined. MCP-1 (A) and MCP-3 (B) levels in organ lysates and serum were quantified by sandwich ELISA. Each bar represents three mice and the experiment was repeated three times. Error bars show SD.

 
MCP-3 production during L. monocytogenes infection requires cytoplasmic invasion by bacteria

Bacterial invasion of host cell cytosol is required for MCP-1 induction by L. monocytogenes (10). To determine whether MCP-3 expression is also induced by an intracellular sensing mechanism, we used an attenuated L. monocytogenes strain that lacks the LLO gene for in vitro and in vivo infections. As shown in Fig. 2A, MCP-3 production was markedly reduced in vivo when the mice were inoculated with LLO-deficient L. monocytogenes or HKL, while infection with ActAko bacteria, which invade the cytosol but are attenuated because they are unable to spread from cell to cell, induced MCP-3. For in vitro analyses, bone marrow-derived macrophages were infected with wild-type (WT) or LLO-deficient L. monocytogenes, or stimulated with HKL. Minimal MCP-3 induction was detected when macrophages were infected with LLO-deficient bacteria or exposed to HKL (Fig. 2B). These experiments, therefore, suggest that MCP-3 and MCP-1 are both induced by cytosol invasion by live L. monocytogenes.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 2. MCP-3 induction requires cytosol invasion by bacteria. A, C57BL/6 mice were infected i.v. with 3000 WT, 108 LLO-deficient L. monocytogenes, 5 x 105 ActA-deficient L. monocytogenes, or immunized with 109 HKL. Spleens were removed and lysed 24 h later. MCP-1 and MCP-3 levels in spleen lysates were quantified by ELISA. Each bar represents three mice and the experiment was repeated twice. Error bars show SD. B, Bone marrow-derived macrophages from C57BL/6 mice were infected with WT, LLO-deficient L. monocytogenes strains at a 5:1 ratio or cultured with 108/ml HKL. As a positive control, cells were cultured with 100 ng/ml LPS. Supernatants were collected at indicated time, and chemokine levels were quantified by ELISA. Each bar represents the average of three wells, and this experiment was repeated twice.

 
MCP-3 is a functional CCR2 ligand during in vivo L. monocytogenes infection

CCR2 signaling is pivotal for innate immune defense against L. monocytogenes infection. Although several MCPs can bind to CCR2, MCP-1 and MCP-3 were shown to be major ligands for CCR2 in inflammatory settings (39). To examine the role of MCP-3 in innate immune defense against bacterial infection, we compared susceptibilities of WT C57BL/6 mice and mice lacking CCR2, MCP-1, or MCP-3 to L. monocytogenes infection. Fig. 3A demonstrates that MCP-1-, MCP-3-, and CCR2-deficient mice had increased numbers of L. monocytogenes in spleens 3 days following bacterial inoculation when compared with WT mice. The increased susceptibility of MCP-1-, MCP-3-, or CCR2-deficient mice was even more apparent 5 days following infection (Fig. 3B). Between days 3 and 5 of infection in WT mice, the number of viable bacteria decreased by 80%, while the number of bacteria in MCP-1–/– and MCP-3–/– mice remained similar during this period. In contrast, the number of bacteria in spleens of CCR2–/– mice increased 500-fold between days 3 and 5 of infection. The similar intermediate phenotype of MCP-1–/– and MCP-3–/– mice indicates that MCP-1 and MCP-3 both contribute to innate immune defense against L. monocytogenes infection.


Figure 3
View larger version (7K):
[in this window]
[in a new window]

  		FIGURE 3. MCP-3–/– mice are more susceptible to L. monocytogenes infection. C57BL/6, MCP-1–/–, MCP-3–/–, and CCR2–/– mice were infected i.v. with 3000 L. monocytogenes. Spleens were removed at (A) day 3 and (B) day 5, and viable bacteria were quantified. Mean numbers of CFUs from groups of 5–10 mice are shown. Error bars represent SD, and this experiment was repeated twice. **, p < 0.01 as compared with WT controls. Double dagger ({ddagger}) indicates the number of dead animals/total in the group.

 
MCP-3 is required to maintain homeostatic levels of circulating inflammatory monocytes

The frequency of circulating inflammatory monocytes is diminished in CCR2-deficient mice. The extent to which MCP-1 and MCP-3 contribute to inflammatory monocyte emigration from bone marrow in the absence of infection is unclear. Therefore, we determined the frequency of inflammatory monocytes in spleen, blood, and bone marrow of naive WT, MCP-1–/–, MCP-3–/–, and CCR2–/– mice. We identified inflammatory monocytes by their high level expression of Ly6C and intermediate to high level expression of CD11b. In Fig. 4, flow cytometric analysis revealed that the percentage of CD11bintLy6Chigh cells was significantly reduced in spleens (Fig. 4A) and blood (Fig. 4B), and that it was retained in the bone marrow (Fig. 4C) of naive MCP-1–/–, MCP-3–/–, and CCR2–/– mice. Compared with MCP-1–/– mice, MCP-3–/– mice had a greater reduction of circulating monocytes, suggesting that MCP-3 may, under noninflammatory circumstances, play a more important role in maintaining monocyte homeostasis.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 4. Homeostatic monocyte trafficking is impaired in the absence of MCP-3. Spleen (A), blood (B), and bone marrow (C) were harvested from naive C57BL/6, MCP-1–/–, MCP-3–/–, and CCR2–/– mice. Cells were stained for CD11b and Ly6C. A large gate was drawn on the live lymphocyte/monocyte population, and expression of CD11b and Ly6C was analyzed. Representative dot plots for three mice per group are shown on left. Percentages of CD11bintLy6Chigh cells in naive WT, MCP-1–/–, MCP-3–/–, and CCR2–/– spleens and circulation are shown on right. Each bar represents the average of six to nine mice from two independent experiments. Error bars represent SD. *, p = 0.012; **, p < 0.01; and ***, p < 0.0001 as compared with WT controls.

 
Defective Tip-DC recruitment in MCP-3–/– mice following infection

Previous studies characterized a novel dendritic cell subset, Tip-DCs, which are recruited to the spleen in a CCR2-dependent manner and which serve as a major source of TNF and iNOS during early L. monocytogenes infection (35). To determine whether the increased susceptibility of MCP-3–/– mice correlates with a defect in Tip-DC recruitment following infection, we examined Tip-DC populations in spleens of MCP-3–/– mice. The level of CD11bintLy6Chigh Tip-DCs is markedly decreased in the spleens (Fig. 5A) and blood (Fig. 5B) of infected MCP-1–/–, MCP-3–/–, and CCR2–/– mice. The magnitude of the decrease in Tip-DC recruitment correlates with the increased susceptibility of mice to L. monocytogenes infection, with intermediate numbers of TipDCs in MCP-1–/– and MCP-3–/– mice compared with WT and CCR2–/– mice. These results suggest that both MCP-1 and MCP-3 play a partial role in monocyte trafficking during infection. Thus, MCP-1-independent monocyte recruitment is likely mediated by MCP-3 signaling through CCR2, while MCP-3-independent recruitment is likely mediated by MCP-1. However, an MCP-1–/–MCP-3–/– mouse strain is not currently available, and thus it is not possible to determine whether MCP-1 and MCP-3 account for all CCR2-dependent inflammatory monocyte recruitment.


Figure 5
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 5. MCP-3-mediated recruitment of Tip-DCs following infection. Spleens (A), blood (B), and bone marrows (C) were harvested from C57BL/6, MCP-1–/–, MCP-3–/–, and CCR2–/– mice infected i.v. with 3000 L. monocytogenes 24 and 48 h postinfection. Cells were stained for CD11b and Ly6C. A large gate was drawn on the live lymphocyte/monocyte population, and expression of CD11b and Ly6C was analyzed. Representative dot plots for three mice per group are shown on the left. Percentages of CD11bintLy6Chigh cells in infected WT, MCP-1–/–, MCP-3–/–, and CCR2–/– spleens, blood, and bone marrow are shown on the right. D–F, Kinetics of CD11bintLy6Chigh population in WT, MCP-1–/–, MCP-3–/–, and CCR2–/– spleens (D), blood (E), and bone marrows (F) following infection are shown. Each bar or time point represents the average of 9–12 mice from three independent experiments. Error bars represent SD. *, p = 0.02; ***, p < 0.0001 as compared with WT controls.

 
Retention of monocyte population in bone marrow of MCP-3–/– mice following infection

Reduction in Tip-DC recruitment to the spleen and blood may result from the failure of monocytes to emigrate from the bone marrow or from abnormal trafficking of monocytes to other tissues. Recent studies demonstrated that CCR2-mediated signals are required for inflammatory monocyte emigration from bone marrow into the circulation (11). To determine whether MCP-3 is required for monocyte emigration from the bone marrow in the setting of L. monocytogenes infection, we examined the CD11bintLy6Chigh monocyte population in bone marrow of WT, MCP-1–/–, MCP-3–/–, and CCR2–/– mice 24 h following infection. We detected an increase in CD11bintLy6Chigh cells in the bone marrow of MCP-1–/–, MCP-3–/–, and CCR2–/– mice (Fig. 5C). Similar to the observation we had in naive mice, MCP-3–/– mice had a more severe defect in monocyte emigration from bone marrow during early L. monocytogenes infection than did MCP-1–/– mice. Defects in monocyte recruitment in MCP-1–/– and MCP-3–/– mice were most dramatic during early infection. Differences in monocyte frequencies between WT and MCP-1–/– or MCP-3–/– mice, although significant, were diminished 48 h after infection in spleen (Fig. 5D), blood (Fig. 5E), and bone marrow (Fig. 5F).

Normal development of Tip-DCs in MCP-3–/–-infected spleen

To understand whether MCP-1 and MCP-3 have roles in activation in addition to recruitment of inflammatory monocytes, we examined TNF-{alpha} and iNOS expression by inflammatory monocyte population in the spleen. Intracellular cytokine staining results showed that TNF-{alpha} (Fig. 6A) and iNOS (Fig. 6B) expression levels in CD11bintLy6Chigh cells in MCP-1–/– and MCP-3–/– spleens were similar to WT mice, indicating MCP-1 and MCP-3 are not required for Tip-DC activation. Consistent with results we obtained by CD11b and Ly6C surface staining, we also observed diminished recruitment of CD11bintTNF-{alpha}high and CD11bintiNOS+ cells in MCP-1–/–, MCP-3–/–, and CCR2–/– mice. These results suggest that higher susceptibilities of MCP-1–/– and MCP-3–/– mice are due to decreased recruitment of inflammatory monocytes rather than impaired differentiation of recruited monocytes into Tip-DCs.


Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 6. MCP-3 is not required for Tip-DC development. Splenocytes from C57BL/6, MCP-1–/–, MCP-3–/–, and CCR2–/– mice infected with 3000 L. monocytogenes were collected 24 h postinfection for intracellular TNF staining and 30 h postinfection for intracellular iNOS staining. Cells were gated on lymphocytes/monocytes, and expression of CD11b and TNF-{alpha} in the gate was analyzed. The percentages of (A) CD11bintTNF-{alpha}high cells and (B) CD11bintiNOS+ cells are indicated. Representative dot plots for three mice per group are shown. The experiment was repeated twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recruitment and targeting of inflammatory cells during infection is a complex process that is enabled, in part, by chemokine-mediated signals. The aggregate inflammatory response depends on recruitment of many different cell types that either circulate in the bloodstream or reside in depots throughout the body, such as spleen, lymph nodes, and bone marrow. Among the cytokines and receptors that are available to coordinate this process are the roughly 50 distinct chemokines and 20 different chemokine receptors encoded in the mammalian genome. Some chemokine-chemokine receptor pairs are monogamous, such as CXCL12-CXCR4 and CCL20-CCR6. Other chemokines, however, bind to multiple receptors, such as RANTES (CCL5) with CCR1, CCR3, and CCR5. In contrast, a number of chemokine receptors bind multiple chemokine ligands, as demonstrated by the association of CCR7 with CCL19 and CCL21, and the response of CCR2 to MCP-1, MCP-2, MCP-3, and MCP-5. Although CCR2 responds to multiple ligands in vitro, the relative contribution of each ligand to cellular trafficking and immune defense during bacterial infection has remained largely unexplored.

In this study, we show similar induction kinetics and expression levels of two CCR2 ligands, MCP-1 and MCP-3, following L. monocytogenes infection. Expression of either MCP-1 or MCP-3 depends on bacterial invasion of the host cell cytosol. Both MCP-1 and MCP-3, under uninflamed conditions, promote homeostatic emigration of inflammatory monocytes from bone marrow into circulating blood. We demonstrate that MCP-1 and MCP-3 both contribute to emigration of monocytes from the bone marrow and Tip-DC recruitment into the spleen during L. monocytogenes infection. Lack of either chemokine results in impaired clearance of bacteria from the spleen and enhanced susceptibility to infection.

Inflammatory chemokines are rapidly induced following infection, and they bind to their receptors to initiate cellular migration and activation. Recruiting inflammatory cells expeditiously and directing them to sites of infection so they can kill invading microbes is a core responsibility of the innate immune system. Precise regulation of chemokine expression, receptor association, and subsequent cellular responses likely optimizes protection and minimizes deleterious effects of uncontrolled microbial growth and invasion. It remains unclear, however, how expression of multiple chemokines that bind the same receptor contributes to innate immune defense. A simple hypothesis is that expression of several ligands for a critical receptor provides redundancy in the event that one ligand is either lost or inactivated. In this scenario, the function of these different ligands would be essentially identical. An alternative but more complex model is that different chemokines binding the same receptor are not simply redundant but provide distinct signals, perhaps due to variations in their association with the chemokine receptor, their ability to dimerize/oligomerize, or their association with GAGs. In this setting, different chemokines would provide distinct information to responding cells, either at the signaling level or at a spatial level, depending on where the chemokine binds. Some evidence in support of the second model comes from studies of CCR7-mediated cell activation and recruitment. Although CCL19 and CCL21 both bind CCR7, they provide distinct signals to responding cells, as detected by in vitro disparities in receptor desensitization, receptor phosphorylation, and ERK1/2 activation (42). In vivo, CCL21 is sufficient for the proper organization of lymphoid T cell zones and migration of lymphocytes, including transmigration across high endothelial venules, while CCL19, but not CCL21, contributes to homeostatic maintenance of T cells (43).

In our study, we observed similar defects in monocyte trafficking and similarly increased CFUs in MCP-1–/– and MCP-3–/– mice following L. monocytogenes infection. In comparison to CCR2–/– mice, MCP-1–/– and MCP-3–/– mice have intermediate phenotypes, and thus MCP-1 and MCP-3 appear to contribute nearly equivalently to monocyte recruitment and antimicrobial clearance. It is possible that MCP-1 and MCP-3 make purely redundant contributions to monocyte recruitment. In support of this scenario, our studies find similar kinetics of MCP-1 and MCP-3 induction and roughly similar amounts of chemokine in most tissues. In contrast, our studies also detected subtle differences in the duration of MCP-1 and MCP-3 expression in tissues and a marked increase of MCP-3, but not MCP-1, protein levels in kidneys of infected mice. Whether these differences in chemokine levels have functional implications will require further study. Additionally, it is still possible other CCR2 ligands such as MCP-2 and MCP-5 play a role in the monocyte recruitment process following infection, although they are not implicated in homeostatic monocyte emigration (39).

Characterization of human MCP-1 and MCP-3 demonstrated differences between these two chemokines that might affect their activities in vivo. Human MCP-3, for example, binds to heparan or heparan sulfate as a monomer, while human MCP-1 only associates as a dimer (44, 45). If MCP-1 and MCP-3 make distinct contributions to monocyte recruitment, it is possible that these two chemokines function in parallel, with each controlling the movement of a subset of monocytes or, in series, with each controlling the movement of the whole monocyte population at distinct stages of their migration from bone marrow to the site of infection. Although our results indicate that MCP-1 and MCP-3 both function at the level of monocyte emigration from the bone marrow, because the frequencies of monocytes in the bone marrow of MCP-1–/– and MCP-3–/– mice were similar, neither the "in parallel" or "in series" model can be excluded. Thus, it is possible that two populations of monocytes reside in the bone marrow, one responding to MCP-1 and the other to MCP-3 (Fig. 7A). Alternatively, it is possible that MCP-1 directs the movement of monocytes from one location in the bone marrow to another, and that MCP-3 provides an additional stimulus that enhances movement into the circulation (Fig. 7B). Distinguishing between these models will likely require intravital studies of monocyte trafficking within bone marrow.


Figure 7
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 7. Models of MCP-1 and MCP-3 functions in monocyte recruitment process. A, Parallel model. Two populations of monocytes reside in different bone marrow niches; one responds to MCP-1 and the other responds to MCP-3. MCP-1 and MCP-3 are each responsible for one population of monocyte emigration from the bone marrow. B, Series model. Monocyte emigration from bone marrow consists of an inner bone marrow mobilization step and a bone marrow-to-circulation step. MCP-1 and MCP-3 each contributes to one step of this two-step process.

 
In summary, MCP-1 and MCP-3 have additive roles in monocyte recruitment and host defense against L. monocytogenes infection. Low secretion of MCP-1 and MCP-3 under normal conditions maintains homeostatic migration of inflammatory monocytes from bone marrow into the circulation. During infection, this process is enhanced by high levels of MCP-1 and MCP-3 in the spleen and serum, or by local production of these two chemokines in the bone marrow. It is still not clear where MCP-1 and MCP-3 are produced and where they establish chemokine gradients in vivo following infection. Further investigation of the expression and activity of these two chemokines during infection will be valuable to dissect the respective roles of these two CCR2 ligands.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 grants (R37AI03903 to E.G.P. and R01 HL52773 and R01 HL63894 to I.F.C.). Back

2 Address correspondence and reprint requests to Dr. Eric G. Pamer, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 9, New York, NY 10021. E-mail address: pamere{at}mskcc.org Back

3 Abbreviations used in this paper: DC, dendritic cell; GAG, glycosaminoglycan; HKL, heat-killed Listeria monocytogenes; iNOS, inducible NO synthase; LLO, listeriolysin O; Tip-DC, TNF- and iNOS-producing DC; WT, wild type. Back

Received for publication December 17, 2007. Accepted for publication March 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Rot, A., U. H. von Andrian. 2004. Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. Annu. Rev. Immunol. 22: 891-928. [Medline]
  2. Allen, S. J., S. E. Crown, T. M. Handel. 2007. Chemokine: receptor structure, interactions, and antagonism. Annu. Rev. Immunol. 25: 787-820. [Medline]
  3. Kurihara, T., G. Warr, J. Loy, R. Bravo. 1997. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor. J. Exp. Med. 186: 1757-1762. [Abstract/Free Full Text]
  4. Lu, B., B. J. Rutledge, L. Gu, J. Fiorillo, N. W. Lukacs, S. L. Kunkel, R. North, C. Gerard, B. J. Rollins. 1998. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J. Exp. Med. 187: 601-608. [Abstract/Free Full Text]
  5. Peters, W., H. M. Scott, H. F. Chambers, J. L. Flynn, I. F. Charo, J. D. Ernst. 2001. Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 98: 7958-7963. [Abstract/Free Full Text]
  6. Robben, P. M., M. LaRegina, W. A. Kuziel, L. D. Sibley. 2005. Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis. J. Exp. Med. 201: 1761-1769. [Abstract/Free Full Text]
  7. Boring, L., J. Gosling, S. W. Chensue, S. L. Kunkel, R. V. Farese, Jr, H. E. Broxmeyer, I. F. Charo. 1997. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J. Clin. Invest. 100: 2552-2561. [Medline]
  8. Boring, L., J. Gosling, M. Cleary, I. F. Charo. 1998. Decreased lesion formation in CCR2–/– mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 394: 894-897. [Medline]
  9. Huang, D. R., J. Wang, P. Kivisakk, B. J. Rollins, R. M. Ransohoff. 2001. Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis. J. Exp. Med. 193: 713-726. [Abstract/Free Full Text]
  10. Serbina, N. V., W. Kuziel, R. Flavell, S. Akira, B. Rollins, E. G. Pamer. 2003. Sequential MyD88-independent and -dependent activation of innate immune responses to intracellular bacterial infection. Immunity 19: 891-901. [Medline]
  11. Serbina, N. V., E. G. Pamer. 2006. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat. Immunol. 7: 311-317. [Medline]
  12. Lesnik, P., C. A. Haskell, I. F. Charo. 2003. Decreased atherosclerosis in CX3CR1–/– mice reveals a role for fractalkine in atherogenesis. J. Clin. Invest. 111: 333-340. [Medline]
  13. Tacke, F., D. Alvarez, T. J. Kaplan, C. Jakubzick, R. Spanbroek, J. Llodra, A. Garin, J. Liu, M. Mack, N. van Rooijen, et al 2007. Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques. J. Clin. Invest. 117: 185-194. [Medline]
  14. Hokeness, K. L., W. A. Kuziel, C. A. Biron, T. P. Salazar-Mather. 2005. Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-{alpha}β-induced inflammatory responses and antiviral defense in liver. J. Immunol. 174: 1549-1556. [Abstract/Free Full Text]
  15. Jiang, D., J. Liang, J. Hodge, B. Lu, Z. Zhu, S. Yu, J. Fan, Y. Gao, Z. Yin, R. Homer, et al 2004. Regulation of pulmonary fibrosis by chemokine receptor CXCR3. J. Clin. Invest. 114: 291-299. [Medline]
  16. Khan, I. A., S. Y. Thomas, M. M. Moretto, F. S. Lee, S. A. Islam, C. Combe, J. D. Schwartzman, A. D. Luster. 2006. CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection. PLoS Pathog. 2: e49[Medline]
  17. Lavergne, E., B. Combadiere, O. Bonduelle, M. Iga, J. L. Gao, M. Maho, A. Boissonnas, P. M. Murphy, P. Debre, C. Combadiere. 2003. Fractalkine mediates natural killer-dependent antitumor responses in vivo. Cancer Res. 63: 7468-7474. [Abstract/Free Full Text]
  18. Thapa, M., W. A. Kuziel, D. J. Carr. 2007. Susceptibility of CCR5-deficient mice to genital herpes simplex virus type 2 is linked to NK cell mobilization. J. Virol. 81: 3704-3713. [Abstract/Free Full Text]
  19. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99: 23-33. [Medline]
  20. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, U. H. von Andrian. 2000. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J. Exp. Med. 191: 61-76. [Abstract/Free Full Text]
  21. Ohl, L., M. Mohaupt, N. Czeloth, G. Hintzen, Z. Kiafard, J. Zwirner, T. Blankenstein, G. Henning, R. Forster. 2004. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity 21: 279-288. [Medline]
  22. Kaiser, A., E. Donnadieu, J. P. Abastado, A. Trautmann, A. Nardin. 2005. CC chemokine ligand 19 secreted by mature dendritic cells increases naive T cell scanning behavior and their response to rare cognate antigen. J. Immunol. 175: 2349-2356. [Abstract/Free Full Text]
  23. Worbs, T., T. R. Mempel, J. Bolter, U. H. von Andrian, R. Forster. 2007. CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo. J. Exp. Med. 204: 489-495. [Abstract/Free Full Text]
  24. Hardtke, S., L. Ohl, R. Forster. 2005. Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B-cell help. Blood 106: 1924-1931. [Abstract/Free Full Text]
  25. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Forster, J. G. Cyster. 2002. Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position. Nature 416: 94-99. [Medline]
  26. Hugues, S., A. Scholer, A. Boissonnas, A. Nussbaum, C. Combadiere, S. Amigorena, L. Fetler. 2007. Dynamic imaging of chemokine-dependent CD8+ T cell help for CD8+ T cell responses. Nat. Immunol. 8: 921-930. [Medline]
  27. Grabovsky, V., S. Feigelson, C. Chen, D. A. Bleijs, A. Peled, G. Cinamon, F. Baleux, F. Arenzana-Seisdedos, T. Lapidot, Y. van Kooyk, et al 2000. Subsecond induction of {alpha}4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192: 495-506. [Abstract/Free Full Text]
  28. Lau, E. K., C. D. Paavola, Z. Johnson, J. P. Gaudry, E. Geretti, F. Borlat, A. J. Kungl, A. E. Proudfoot, T. M. Handel. 2004. Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo. J. Biol. Chem. 279: 22294-22305. [Abstract/Free Full Text]
  29. Proudfoot, A. E., T. M. Handel, Z. Johnson, E. K. Lau, P. LiWang, I. Clark-Lewis, F. Borlat, T. N. Wells, M. H. Kosco-Vilbois. 2003. Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. USA 100: 1885-1890. [Abstract/Free Full Text]
  30. Witt, D. P., A. D. Lander. 1994. Differential binding of chemokines to glycosaminoglycan subpopulations. Curr. Biol. 4: 394-400. [Medline]
  31. Czaplewski, L. G., J. McKeating, C. J. Craven, L. D. Higgins, V. Appay, A. Brown, T. Dudgeon, L. A. Howard, T. Meyers, J. Owen, et al 1999. Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1{alpha}, MIP-1β, and RANTES: characterization of active disaggregated chemokine variants. J. Biol. Chem. 274: 16077-16084. [Abstract/Free Full Text]
  32. Woolf, E., I. Grigorova, A. Sagiv, V. Grabovsky, S. W. Feigelson, Z. Shulman, T. Hartmann, M. Sixt, J. G. Cyster, R. Alon. 2007. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nat. Immunol. 8: 1076-1085. [Medline]
  33. Gellin, B. G., C. V. Broome. 1989. Listeriosis. J. Am. Med. Assoc. 261: 1313-1320. [Abstract/Free Full Text]
  34. Bielecki, J., P. Youngman, P. Connelly, D. A. Portnoy. 1990. Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature 345: 175-176. [Medline]
  35. Serbina, N. V., T. P. Salazar-Mather, C. A. Biron, W. A. Kuziel, E. G. Pamer. 2003. TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection. Immunity 19: 59-70. [Medline]
  36. Van Damme, J., P. Proost, J. P. Lenaerts, G. Opdenakker. 1992. Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J. Exp. Med. 176: 59-65. [Abstract/Free Full Text]
  37. Taub, D. D., P. Proost, W. J. Murphy, M. Anver, D. L. Longo, J. van Damme, J. J. Oppenheim. 1995. Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes. J. Clin. Invest. 95: 1370-1376. [Medline]
  38. Allavena, P., G. Bianchi, D. Zhou, J. van Damme, P. Jilek, S. Sozzani, A. Mantovani. 1994. Induction of natural killer cell migration by monocyte chemotactic protein-1, -2 and -3. Eur. J. Immunol. 24: 3233-3236. [Medline]
  39. Tsou, C. L., W. Peters, Y. Si, S. Slaymaker, A. M. Aslanian, S. P. Weisberg, M. Mack, I. F. Charo. 2007. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J. Clin. Invest. 117: 902-909. [Medline]
  40. Kuziel, W. A., S. J. Morgan, T. C. Dawson, S. Griffin, O. Smithies, K. Ley, N. Maeda. 1997. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc. Natl. Acad. Sci. USA 94: 12053-12058. [Abstract/Free Full Text]
  41. Lauvau, G., S. Vijh, P. Kong, T. Horng, K. Kerksiek, N. Serbina, R. A. Tuma, E. G. Pamer. 2001. Priming of memory but not effector CD8 T cells by a killed bacterial vaccine. Science 294: 1735-1739. [Abstract/Free Full Text]
  42. Kohout, T. A., S. L. Nicholas, S. J. Perry, G. Reinhart, S. Junger, R. S. Struthers. 2004. Differential desensitization, receptor phosphorylation, β-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7. J. Biol. Chem. 279: 23214-23222. [Abstract/Free Full Text]
  43. Link, A., T. K. Vogt, S. Favre, M. R. Britschgi, H. Acha-Orbea, B. Hinz, J. G. Cyster, S. A. Luther. 2007. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8: 1255-1265. [Medline]
  44. Schenauer, M. R., Y. Yu, M. D. Sweeney, J. A. Leary. 2007. CCR2 chemokines bind selectively to acetylated heparan sulfate octasaccharides. J. Biol. Chem. 282: 25182-25188. [Abstract/Free Full Text]
  45. Yu, Y., M. D. Sweeney, O. M. Saad, S. E. Crown, A. R. Hsu, T. M. Handel, J. A. Leary. 2005. Chemokine-glycosaminoglycan binding: specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry. J. Biol. Chem. 280: 32200-32208. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
P. Y. Lee, Y. Li, Y. Kumagai, Y. Xu, J. S. Weinstein, E. S. Kellner, D. C. Nacionales, E. J. Butfiloski, N. van Rooijen, S. Akira, et al.
Type I Interferon Modulates Monocyte Recruitment and Maturation in Chronic Inflammation
Am. J. Pathol., November 1, 2009; 175(5): 2023 - 2033.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Montfort, B. de Badts, V. Douin-Echinard, P. G. P. Martin, J. Iacovoni, C. Nevoit, N. Therville, V. Garcia, M.-A. Bertrand, M.-H. Bessieres, et al.
FAN Stimulates TNF{alpha}-Induced Gene Expression, Leukocyte Recruitment, and Humoral Response
J. Immunol., October 15, 2009; 183(8): 5369 - 5378.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Takahashi, C. Galligan, L. Tessarollo, and T. Yoshimura
Monocyte Chemoattractant Protein-1 (MCP-1), Not MCP-3, Is the Primary Chemokine Required for Monocyte Recruitment in Mouse Peritonitis Induced with Thioglycollate or Zymosan A
J. Immunol., September 1, 2009; 183(5): 3463 - 3471.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. J. Crane, K. L. Hokeness-Antonelli, and T. P. Salazar-Mather
Regulation of Inflammatory Monocyte/Macrophage Recruitment from the Bone Marrow during Murine Cytomegalovirus Infection: Role for Type I Interferons in Localized Induction of CCR2 Ligands
J. Immunol., August 15, 2009; 183(4): 2810 - 2817.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. A. Szymczak and G. S. Deepe Jr
The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity
J. Immunol., August 1, 2009; 183(3): 1964 - 1974.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. V. Serbina, T. M. Hohl, M. Cherny, and E. G. Pamer
Selective Expansion of the Monocytic Lineage Directed by Bacterial Infection
J. Immunol., August 1, 2009; 183(3): 1900 - 1910.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Jia, I. Leiner, G. Dorothee, K. Brandl, and E. G. Pamer
MyD88 and Type I Interferon Receptor-Mediated Chemokine Induction and Monocyte Recruitment during Listeria monocytogenes Infection
J. Immunol., July 15, 2009; 183(2): 1271 - 1278.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jia, T.
Right arrow Articles by Pamer, E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jia, T.
Right arrow Articles by Pamer, E. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS