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Originally published online as doi:10.1189/jlb.1202613 on November 3, 2003

Published online before print November 3, 2003
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(Journal of Leukocyte Biology. 2004;75:275-285.)
© 2004 by Society for Leukocyte Biology

Comparative evaluation of CC chemokine-induced migration of murine CD8{alpha}+ and CD8{alpha}- dendritic cells and their in vivo trafficking

Bridget L. Colvin*,{dagger}, Adrian E. Morelli*,{ddagger}, Alison J. Logar*, Audrey H. Lau*,{dagger} and Angus W. Thomson*,{dagger},{ddagger},1

* Thomas E. Starzl Transplantation Institute and Departments of
{ddagger} Surgery and
{dagger} Immunology, University of Pittsburgh Medical Center, Pennsylvania

1 Correspondence: W1544 Biomedical Science Tower, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213. E-mail: thomsonaw{at}msx.upmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Murine CD11c+CD8{alpha}- and CD11c+CD8{alpha}+ dendritic cells (DCs) differentially regulate T cell responses. Although specific chemokines that recruit immature (i) or mature (m) CD8{alpha}- DCs have been identified, little is known about the influence of chemokines on CD8{alpha}+ DCs. iDCs and mDCs isolated from spleens of fms-like tyrosine kinase 3 ligand-treated B10 mice were compared directly for migratory responses to a panel of CC chemokines or following local or systemic administration. In vitro assays were performed using Transwell® chambers. iDCs did not respond to any CC chemokines tested. Both subsets of mDCs migrated to CCL19 and CCL21, with consistently lower percentages of CD8{alpha}+ DCs migrating. Chemokine receptor mRNA and protein expression were analyzed, but no correlation between expression and function was demonstrated. In vivo trafficking of fluorochrome-labeled DCs (B10; H2b) was assessed by immunohistochemistry and by rare-event flow cytometric analysis of allogeneic recipient (BALB/c; H2d) draining lymph node (DLN) and spleen cells. Twenty-four hours after intravenous injection, chloromethylfluorescein diacetate-positive CD8{alpha}+ and CD8{alpha}- mDCs were detected by immunohistochemistry in spleens in similar numbers (that decreased over time). Following subcutaneous injection, both DC subsets were detected in DLN at 24 h, but only CD8{alpha}- DCs were evident by flow analysis at 48 h. Although CD8{alpha}+ DCs migrate from peripheral tissues to T cell areas of (allogeneic) secondary lymphoid organs, they appear to mobilize as mDCs and less efficiently than CD8{alpha}- mDCs.

Key Words: leukocyte chemotaxis • antigen-presenting cell • lymphoid tissue


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immunobiology of "lymphoid-related" murine dendritic cells (DCs), characterized by their dual expression of CD11c and the CD8{alpha} homodimer [1 2 3 4 ], has attracted much attention since these cells were first described [5 6 7 8 ]. Early reports suggested that CD8{alpha}+ DCs might be specialized for tolerance induction, as unlike their so-called "myeloid" counterparts (CD11c+CD8{alpha}-), they suppressed CD4+ and CD8+ T cell proliferation [9 , 10 ] and enhanced the apoptotic death of activated T cells [11 ]. Several groups have reported diverse functions for these cells, including selective T helper (Th) cell subset activation in vivo in response to soluble (ovalbumin, keyhole limpet hemocyanin) or viral antigens (Ags) [12 13 14 15 ], suppression of CD8{alpha}- DC-induced delayed-type hypersensitivity to tumor/self-peptide [16 , 17 ], and prolongation of organ allograft survival [4 ]. As with classic myeloid DC, the ability of these Ag-presenting cells to affect immune reactivity also depends on their tissue distribution and migratory properties, both of which are influenced by chemokines.

The in vitro chemotactic responses of immature (i) and mature (m) CD8{alpha}- DCs derived from murine bone marrow (BM) [18 ], skin [19 ], or lymph node (LN) [19 ] from skin [20 , 21 ] or blood in humans [22 23 24 25 ] and from blood in nonhuman primates [26 ] have been well documented. In vivo studies have been much more circumspect [26 , 27 ]. The few reports regarding CD8{alpha}+ DCs have examined only their in vivo trafficking [12 , 28 , 29 ] or the in vitro migration of these DCs [isolated from intestinal Peyer’s patches (PPDCs)] in response to a restricted set of chemokines {CCL19 [macrophage inflammatory protein (MIP)-3ß], CCL20 (MIP-3{alpha}), and CCL21 [secondary lymphoid chemokine (SLC)]} [30 ] and have not adequately considered all the factors that might influence their migration. In particular, the relationship between maturation status and migratory potential of murine DCs in vivo has not been resolved—a discrepancy underlined by the fact that in nonhuman primates, iDCs and mDCs migrate with equal efficiency to draining LN (DLN) after intradermal (i.d.) injection (despite the fact that rhesus mDCs express CC chemokine receptor (CCR)7, and iDCs do not) [26 ]. It is likely that differences in experimental protocol for migration analysis in vivo in the mouse are responsible for the disparate findings. Two groups have reported that spleen-derived, subcutaneous (s.c.)-injected CD8{alpha}+ iDCs fail to migrate to DLN [12 , 29 ], whereas another has observed reduced (as compared with CD8{alpha}- DCs) CD8{alpha}+ iDC migration to the spleen after their intravenous (i.v.) injection [12 ]. O’Connell et al. [31 ] found that in vivo-mobilized, liver-derived CD8{alpha}+ mDCs trafficked to DLN and spleen after s.c. injection, and Drake et al. [28 ] detected in vivo-mobilized, spleen-derived CD8{alpha}+ mDCs in DLN after their s.c. injection in a mixed population together with CD8{alpha}- mDCs. These inconsistencies have not been explained.

Leukocyte recruitment in vivo is mediated largely by chemokines. Each of the four families of chemokines (C, CC, CXC, and CX3C) attracts specific leukocytes [32 33 34 35 36 37 38 39 40 ]. Inducible CC chemokines recruit iDCs to inflamed peripheral tissues, and constitutive CC chemokines attract mDCs to secondary lymphoid organs. There is also recent evidence that chemokines within organ allografts may influence the outcome of transplant survival [41 42 43 44 45 46 ]. In this context, it appears that chemokines elicit migration of cells important in facilitating rejection (i.e., iDCs or alloAg-specific T cells); conversely, chemokines may be able to recruit cells for suppression of rejection (i.e., regulatory T cells or "tolerogenic" DCs). Manipulation/targeting of those chemokines may promote graft survival. In this comparative study, we have analyzed factors that influence trafficking of defined DC subsets in vitro and in vivo. We show for the first time that murine spleen iDCs fail to respond to the same panel of inducible CC chemokines as has been reported for iBM DCs (iBMDCs), despite a similar expression pattern of mRNA for CCRs [18 , 47 ]. In addition, a direct comparison of the in vivo trafficking of CD8{alpha}+ and CD8{alpha}- iDCs and mDCs shows that their ability to reach secondary lymphoid tissues is affected not only by their maturation status but also by their route of administration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Male C57BL/10J (B10; H2b) and BALB/c (H2d) mice, 8–12 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained in the specific pathogen-free facility of the University of Pittsburgh Medical Center (PA) and were fed Purina rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum.

DC isolation, purification, and maturation
Spleen DCs were isolated as described [4 ]. Briefly, B10 mice were treated with recombinant (r) human hematopoietic growth factor fms-like tyrosine kinase 3 ligand (Flt3L or FL; Immunex, Seattle, WA) for 10 days [10 µg mouse/day intraperitoneally (i.p.)] to increase the number of DCs [48 ]. This approach allowed isolation of a greater number of DCs from fewer mice than isolation of DCs from untreated animals. On day 11, spleens were excised and gently macerated, and red blood cells were lysed. No digestive procedures were used to minimize possible perturbation of cell-surface Ag expression. To generate mDCs, cells were incubated overnight (18 h) in RPMI-1640 (Life Technologies, Gaithersburg, MD) supplemented with antibiotics, 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Inc.), and r mouse granulocyte macrophage-colony stimulating factor (GM-CSF; 1000 U/ml; Schering Plough, Kenilworth, NJ) at 37°C, 5% CO2. mDCs, as described previously [4 ], were CD40hi, CD80hi, CD86hi, and major histocompatibility complex (MHC) IIhi and elicited potent, naïve, allogeneic T cell proliferation. By contrast, iDCs, freshly isolated from spleen cell suspensions, expressed CD40-/lo, CD80-/lo, CD86-/lo, and MHC IIlo and exhibited weak, naïve, T cell allostimulatory capacity [4 ]. BMDCs were generated as described previously [49 ]. Briefly, BM was extracted from the femurs and tibias of FL-treated B10 mice, and iBMDCs were harvested from cultures 1 day after extraction and were defined by the same criteria as spleen iDCs. For enrichment of iDCs and mDCs, 16% or 14.5% w/v metrizamide solution was used, respectively. Bulk DCs were incubated with magnetic beads coupled to anti-CD8{alpha} monoclonal antibody (mAb; Miltenyi Biotec, Auburn, CA) at 4°C for 15 min (according to the manufacturer’s instructions). Half of the bulk DCs was then passed through a positive-selection column (Miltenyi Biotec) to obtain a highly purified population (60–90%) of magnetically labeled DCs; the remainder was passed through a depletion column (Miltenyi Biotec) to select for a highly purified population of unbound DCs (75–95%). Freshly isolated CD8{alpha}- cells were further enriched for CD11c by incubation of depletion column-eluted DCs with magnetic beads, coupled to anti-CD11c mAb at 4° for 15 min, and were then passed through a positive-selection column. iBMDCs were further purified via anti-CDllc mAb bead separation after metrizamide enrichment. DCs were then used for phenotypic and functional analyses. Spleens from non-FL-treated mice were digested with 100 U collagenase, teased apart, and then further digested in 400 U collagenase for two 30-min incubation periods to maximize DC recovery. Bulk spleen cell cultures were then enriched for iDCs and mDCs with metrizamide, as reported for cells from FL-treated mice.

Flow cytometric analyses
Cell-surface phenotypic analysis was performed by flow cytometry using an EPICS Elite ESP analyzer (Beckman Coulter, Hialeah, FL). All flow cytometric experiments were done at 4°C. Leukocytes were first blocked with 10% normal goat serum for 10 min and then stained with mAb for 30 min. Cells stained with the appropriate isotype-matched immunoglobulin (BD PharMingen, San Diego, CA) were used as negative controls. DC purity after immunomagnetic bead-sorting was assessed by determining the expression of CD11c and CD8{alpha} using flow cytometry. Phenotypic analysis of cell-surface markers was performed with the following Abs: Phycoerythrin (PE)-CD11b, -CD40, -CD54, -CCR5, -IAb, -H2k, -CD80, and -CD86 (mAb; BD PharMingen); biotinylated CCR1 (C terminus-reactive), CCR1 (N terminus-reactive), CCR5 (C terminus-reactive), CCR7 (C terminus-reactive; goat anti-mouse polyAb), and secondary fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA); FITC-CCR3 and -CCR6 (mAb; R&D Systems, Minneapolis, MN); and biotinylated CCR4 (donkey anti-mouse polyAb) and secondary FITC-conjugated goat anti-donkey (Capralogics Inc., Hardwick, MA). After staining, the cells were fixed with 4% paraformaldehyde (PFA). For some experiments, CD11c+CD8{alpha}+ and CD11c+CD8{alpha}- iDCs and mDCs were flow-sorted according to their positivity for CD11c and CD8{alpha}. Intracellular staining was performed according to the standard BD PharMingen protocol: After Fc-block with CD16/32 (mAb; BD PharMingen), DCs were surface-stained with PE–CD11c and Cy-chrome CD8{alpha} (mAb; BD PharMingen) for 30 min and then fixed for 20 min with 4% PFA. Cells were then permeablized in the presence of biotinylated CCR1, CCR5, or CCR7 (polyAb; Santa Cruz Biotechnology) for 30 min, washed, and then resuspended in permeablization buffer in the presence of the FITC-conjugated secondary goat anti-donkey polyAb (Santa Cruz Biotechnology). After staining, cells were fixed again with 4% PFA.

Chemokines
Mouse r CC chemokines [monocyte chemoattractant protein (MCP)-1, -3, -5 (CCL2, CCL7, CCL12, respectively), macrophage-derived chemokine (MDC; CCL22), MIP-1{alpha}, -1ß, -1{gamma}, -3{alpha}, -3ß (CCL3, CCL4, CCL9/10, CCL20, CCL19, respectively), regulated upon activation, normal T cell expressed and secreted (RANTES; CCL5), SLC (CCL21), and thymus-expressed chemokine (TECK; CCL25)] were diluted to various concentrations [0, 0.01, 0.1, 1, 10, 100 (nM); R&D Systems] for the chemotaxis assay experiments.

Chemotaxis assays
Assays were performed as described previously [50 ] with minor modifications. Purified CD8{alpha}+ or CD8{alpha}- iDCs or mDCs, iBMDCs, or bulk iDCs or mDCs from non-FL-treated mice (1–4x105) were resuspended in 100 µl 0.5% bovine serum albumin (BSA) RPMI 1640 (no FBS; without chemokine) in Transwells® (5 µm pores for mDCs, 5 or 8 µm pores for iDCs; Costar, Cambridge, MA), were placed in 24-well plates with 600 µl chemokine dilution in 0.5% BSA RPMI 1640 per well, and were incubated for 2 h at 37°C in 5% CO2. The Transwells® were then removed, and migrated DCs were collected from the 24-well plates and enumerated using a Coulter Counter (Beckman Coulter). For accurate comparison between experiments, results were expressed as the percentage of migrated DCs. Migration assays were performed in duplicate; experiments were repeated at least three times.

Calcium flux
Ca++ flux was analyzed as described previously [47 ] with minor modifications. DCs were loaded with 2 µM fura-2/AM (Molecular Probes, Leiden, The Netherlands), incubated for 30 min at 37°C in phosphate-buffered saline (PBS) without Mg++ or Ca++, supplemented with 4 (mM) Probenecid (Sigma-Aldrich, St. Louis, MO) in the dark, washed twice in PBS (BioWhittaker, Walkersville, MD), and resuspended in PBS containing 1.2 M Ca++ and 1.5 M Mg++ at 2–3 x 106 cells/ml. Chemokines were added at a concentration of 10 (nM) at indicated times in a continuously stirred cuvette at 37°C in a Model MS-III spectrofluorimeter (Photon Technology, South Brunswick, NJ). The relative ratio of fluorescence emitted at 510 nm following sequential excitation at 340 and 380 nm was then recorded.

In vivo migration
CD8{alpha}+ and CD8{alpha}- (B10, H2b) DCs were cytoplasmically labeled with the green fluorescent dye chloromethylfluorescein diacetate (CMFDA; Molecular Probes; 15 µM; 45 min at 37°C), according to the manufacturer’s instructions. After two washes, 2–4 x 106 CD8{alpha}+ or CD8{alpha}- DCs were injected i.v. into the lateral tail vein or s.c. into one hind footpad of allogeneic (BALB/c, H2d) recipients. Spleens or popliteal DLN were removed 24, 48, or 72 h after i.v. or s.c. injection. Qualitative analysis of in vivo DC migration was performed by immunohistochemical detection of fluorescent (green) cells, whereas quantification of in vivo DC migration was performed by rare-event flow cytometric analysis of green fluorescent cells as compared with control spleens/DLN from age-matched mice that had not been injected with DCs.

Immunostaining of tissue sections
Tissue samples were embedded in Tissue-Tek OCT (Miles Laboratories, Elkhart, IN), snap-frozen in isopentane (prechilled in liquid N2), and stored at -80°C until use. Cryostat sections (8 µm) were mounted on slides pretreated with Vectabond (Vector Laboratories, Burlingame, CA), air-dried, and fixed in cold 4% PFA for 10 min. Sections were blocked with 5% v/v normal goat serum, followed by avidin-blocking solution (Vector Laboratories). Biotinylated anti-CD3{varepsilon} (T cells), -CD11c (DCs), and -CD19 (B cells) mAb (BD PharMingen) and biotinylated F4/80 (red pulp macrophages) and MOMA-1 (marginal zone metallophillic macrophages) mAb (Bachem Bioscience, King of Prussia, PA), followed by Cy3-streptavidin (Jackson Laboratories), were used to localize T cell, DC, B cell, red pulp, and marginal zone areas, respectively.

RNase protection assay
The RNase protection assay (RPA) was performed as described [51 ]. Briefly, RNA was isolated from ~5 x 106 flow-sorted DCs using a total RNA isolation kit (BD PharMingen). RPA was conducted using the RiboQuant mCR-5 multiprobe template set with antisense RNA probes that target CCR1, -1b, -2, -3, -4, and -5 and the housekeeping genes L32 and glyceraldehyde 3-phosphate dehydrogenase (BD PharMingen). CCR6 and -7 probes were included in a customized RiboQuant multiprobe template set (BD PharMingen). The corresponding antisense RNA probe set was included as molecular weight standards. Mouse RNA and RNA degradation controls were included. Yeast tRNA served as a negative control. Quantification of bands was performed by densitometry followed by assessment using Image QuantNT software (Molecular Dynamics, Sunnyvale, CA). The signals from specific mRNA were normalized to signals from the housekeeping genes run on each lane to adjust for loading differences.

Statistical analysis
Results are expressed as means ± 1 SD. Comparisons between means were performed by ANOVA and then by the Newman-Keuls test. Comparison between two means was performed by the Student’s t-test. A P value <=0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunomagnetic bead separation is an efficient means of sorting spleen CD8{alpha}+ and CD8{alpha}- iDCs and mDCs
Flow sorting typically results in highly purified cell populations (>95%) but at the cost of time and limited cell recovery. Therefore, for the chemotaxis assays, we used freshly isolated (iDCs) or overnight GM-CSF-cultured (mDCs) B10-splenic CD8{alpha}+ and CD8{alpha}- DCs enriched on a 16% (iDCs) or 14.5% (mDCs) metrizamide gradient, followed by immunomagnetic bead sorting, a method recently adopted by other groups for DC subset isolation [12 , 52 ]. Metrizamide separation yielded >60% of CD11c+CD86-/lo iDCs (Fig. 1A and 1E ) and >90% of CD11c+CD86hi mDCs (Fig. 1B and 1F) . Forty percent to 60% of iDCs or mDCs was CD8{alpha}+ (Fig. 1A and 1B) . DCs were further purified by immunomagnetic bead sorting into CD8{alpha}+ (~80% iDCs; >90% mDCs) and CD8{alpha}- (~80% iDCs; ~90% mDCs) populations (Fig. 1C and 1D) . As expected, both subsets of iDCs exhibited iDC morphology, were CD40/80/86-/lo and MHC IIlo, and were poor stimulators of allogeneic, naive T cells in mixed leukocyte reaction, and mDCs of both subsets showed abundant dendritic prolongations, were MHC IIhi and CD40/80/86hi (Fig. 1 and data not shown), and exhibited potent, naïve T cell allostimulatory capacity, as previously reported by our group [4 , 31 ].



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  		Figure 1. Immunomagnetic bead sorting yields ample numbers of highly purified DC populations. (A, B) Freshly isolated or overnight (18 h), GM-CSF-cultured spleen cells from 10-day FL-treated B10 mice were layered on 16% w/v or 14.5% w/v (for iDCs or mDCs, respectively) metrizamide/PBS. (C, D) This CD11c+ DC-enriched population was then immunomagnetic bead-sorted according to CD8{alpha} expression, typically yielding ~80% CD8{alpha}+CD86lo and ~80% CD8{alpha}-CD86- iDCs (C and E) and >90% CD8{alpha}+CD86+ and >90% CD8{alpha}-CD86+ mDCs (D and F). DCs were regarded as iDCs or mDCs according to their CD86 expression (E and F) and allostimulatory ability [4 , 31 ]. Appropriate isotype controls are represented by the open histograms; numbers indicate percentage of cells and mean fluorescence intensity in parentheses.

 
Spleen CD8{alpha}+ and CD8{alpha}- iDCs do not migrate in vitro in response to typical iDC migration-inducing CC chemokines
Various mouse CC chemokines were tested in chemotaxis assays to determine the in vitro migratory response of CD8{alpha}+ and CD8{alpha}- iDCs. Surprisingly, although human and mouse BM- and blood-derived and skin iDCs have been reported to respond to CC chemokines [18 , 22 , 23 , 30 ], neither murine spleen iDCs migrated to any of the CC chemokines tested [CCL2 (MCP-1), CCL3 (MIP-1{alpha}), CCL4 (MIP-1ß), CCL5 (RANTES), CCL7 (MCP-3), CCL9/10 (MIP-1{gamma}), CCL12 (MCP-5), CCL19 (MIP-3ß), CCL20 (MIP-3{alpha}), CCL21 (SLC), CCL22 (MDC), or CCL25 (TECK); >=10% migration above control wells without chemokine; Table 1 , Fig. 2A , and data not shown]. To confirm that our assay was appropriate for analysis of chemotaxis of immunobead-sorted spleen iDC migration, we included BM-derived iDCs as positive controls. The iBMDCs exhibited reproducible migration to CCL4 (MIP-1ß) and CCL5 (RANTES; Fig. 2B ), as reported previously [22 , 38 , 39 , 53 ]. As expected, neither CCL19 (MIP-3ß) nor CCL21 (SLC; both chemoattractants for mDCs) elicited migration of splenic CD8{alpha}+ or CD8{alpha}- iDCs or iBMDCs (Table 1 , Fig. 2A and 2B , and data not shown).


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  		Table 1. Assessment of Migratory Responses of i and m CD8{alpha}+ and CD8{alpha}- DC to CC Chemokines

 


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  		Figure 2. DC migration to specific CC chemokines in vitro is dependent on tissue of origin and state of maturation. Immunobead-sorted (unless otherwise noted) DCs were placed in the upper wells of Transwell® chambers over graded concentrations of CC chemokines. (A) Freshly isolated CD8{alpha}+ or CD8{alpha}- iDCs from non-FL (nonimmunobead-sorted)- or FL-treated spleens failed to migrate to any CC chemokines tested (positive chemotaxis was considered >=10% migration over control without chemokines). (B) iBMDCs migrated to CCL4 and CCL5 but to neither of the constitutively expressed chemokines CCL19 and CCL21 (data not shown). (C) Splenic CD8{alpha}+ or CD8{alpha}- mDCs migrated only to CCL19 and CCL21 (data not shown). To assess whether immunomagnetic beads interfere with DC chemotaxis, the migratory response to CCL19 and CCL21 of negatively selected CD8{alpha}- mDCs (unbeaded) was compared with that of negatively selected CD8{alpha}- mDCs incubated with anti-CD11c magnetic beads. CD8{alpha}- mDCs migrated equally well with or without magnetic bead attachment. Three examples of the 12 CC chemokines tested are shown. Data are from a single experiment representative of at least three performed; each chemokine dilution was tested in duplicate.

 
Spleen CD8{alpha}+ and CD8{alpha}- mDCs migrate in vitro in response to mDC migration-inducing chemokines
The constitutively expressed chemokines CCL19 [by DCs and endothelial cells (ECs) in secondary lymphoid tissues] and CCL21 (by ECs in lymphatics and high endothelial venules) [54 55 56 ] have been reported to induce migration of human and murine mDCs [18 , 19 , 22 , 30 , 53 ]. In keeping with these reports, CD8{alpha}+ and CD8{alpha}- splenic mDCs migrated in response to CCL19 and CCL21 but not to the inflammatory CC chemokines tested [CCL2 (MCP-1), CCL3 (MIP-1{alpha}), CCL4 (MIP-1ß), CCL5 (RANTES), CCL7 (MCP-3), CCL9/10 (MIP-1{gamma}), CCL12 (MCP-5), CCL20 (MIP-3{alpha}), CCL22 (MDC), and CCL25 (TECK); Table 1 , Fig. 2C , and data not shown]. CD8{alpha}+ mDCs consistently migrated in fewer numbers in response to CCL19 and CCL21 compared with CD8{alpha}- DCs (Fig. 2C) . As after cell purification, CD8{alpha}+ DCs (unlike CD8{alpha}- DCs) may have retained magnetic beads on the cell surface, we ascertained whether the presence of magnetic beads on the cell surface would affect CD8{alpha}+ DC migration. As shown in the last panel of Figure 2C , CD8{alpha}- DCs bearing anti-CD11c magnetic beads migrated equally well to CCL19 and CCL21 as flow-sorted CD8{alpha}- DCs. Therefore, the presence of immunomagnetic beads on the cell surface did not affect migration.

To ensure FL mobilization of the spleen DCs was not a factor influencing their ability to migrate, bulk spleen iDC and mDC were isolated from nontreated mice, and their chemotactic responses were tested to a select group of CC chemokines. As it required a large number of mice to isolate enough iDCs for one chemotaxis assay and as only a very few non-FL-treated iDCs could be isolated, even from a large number of spleens, the iDCs were not separated into CD8{alpha}+ and CD8{alpha}- populations. Like iBMDC, whose migration in response to CC chemokines is not affected by exposure to FL [18 , 47 ], spleen iDC from non-FL-treated mice did not migrate to CCL4, CCL19, CCL20, CCL21 (data not shown), or CCL5 (Table 1 and Fig. 2A ), and nontreated spleen mDC migrated to CCL19 and CCL21 (but not CCL4, CCL5, or CCL20) in a similar manner to their FL-mobilized counterparts (Table 1 and data not shown).

CCR mRNA and protein expression in spleen CD8{alpha}+ and CD8{alpha}- DCs do not correlate with (apparent) functional CCR expression
The lack of available cell-surface staining murine CCR Ab coupled with the absence in vitro of migration to inflammatory CC chemokines led us to analyze the pattern of CCR mRNA expressed by splenic DCs using RPA. CD8{alpha}+ and CD8{alpha}- iDCs showed the typical pattern of down-regulation of inflammatory CCR mRNA expression (CCR1, -2, and -5), especially CCR2 upon maturation, and concomitant up-regulation of CCR7 (Fig. 3A and 3B ), was similar to that reported for murine BMDCs (in the presence [18 ] and absence [18 , 47 ] of FL). Despite the expression of mRNA for CCR1, -2, and -5, both spleen iDC subsets failed to migrate in response to any of their ligands (Table 1 and Fig. 2 ), although iBMDCs migrated to the CCR1 and CCR5 ligands CCL4 and CCL5. It is interesting that expression of mRNA for CCR6, a chemokine receptor (CR) typically considered to be expressed on precursor/iDC populations [20 , 21 , 30 ], was low in iDCs but increased in mDCs. We therefore then analyzed untreated spleen CD8{alpha}+ and CD8{alpha}- iDC and mDC mRNA to determine if this unexpected CCR6 expression pattern was a result of FL treatment. i and m spleen CD8{alpha}+ and CD8{alpha}- DCs from non-FL-treated mice expressed an identical pattern of mRNA for CCR 1–7 (data not shown) as compared with all spleen DC subsets from FL-treated mice, indicating that the pattern of CCR6 mRNA was not a result of treatment of the mice with the growth factor. Nevertheless, neither subset at either stage of maturation (with or without FL mobilization) exhibited a positive migratory response to the CCR6 ligand CCL20 (MIP-3{alpha}).



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  		Figure 3. CCR mRNA expression in spleen DCs does not correlate directly with functional CCR protein production. CD8{alpha}+ and CD8{alpha}- iDCs and mDCs were flow-sorted for maximum purity (>95%). (A) Both iDC subsets down-regulated CCR1, CCR2, and CCR5 mRNA expression and up-regulated CCR6 and CCR7 message upon maturation into mDCs. (B) Densitometric analysis for quantitation of cytokine mRNA expression. Analysis of each lane was performed on scanned autoradiographs, and all values are expressed relative to corresponding housekeeping gene transcripts (L32). Data are from one experiment representative of three performed. (C) Intracellular staining of CCR1, -5, and -7 revealed no significant difference in the production of CCR between either subset of either state of maturation. (D) Neither CD8{alpha}+ (left) nor CD8{alpha}- (middle) spleen iDC exhibited a change in intracellular Ca++ after exposure to CCL4 (data not shown) or CCL5, as indicated by a lack of increase in the overall fluorescence ratio after the initial spike in fluorescence that occurs when there is a perturbation in the spectrofluorimeter reading (as occurs with the addition of the chemokine). Ca++ flux was seen after exposure to ionomycin (positive control; left and middle). However, iBMDC (right) responded to CCL5.

 
As CCR mRNA expression did not correlate with spleen iDC migration and as CD8{alpha}+ mDCs migrated in consistently fewer numbers than CD8{alpha}- DCs (possibly indicating lower expression of CCR7), we attempted to measure DC surface CCR protein expression against CCR1, -3, -4, -5, -6, and -7. However, in ~25 experiments, CCR detection was not reproducible. We therefore endeavored to measure intracellular protein production with anti-CCR1, -CCR5, and -CCR7 polyAb. Although these CCRs were detected consistently in iDCs and mDCs of both subsets, no conclusions can be drawn regarding their differential surface expression, as apparent intracellular protein production showed little variation among the cell subtypes analyzed (Fig. 3C) . As iDCs contained mRNA and protein for CCR1 and -5 but did not migrate to any of the ligands for these CRs, we performed a Ca++ flux assay to determine if the DCs expressed CRs that could be activated, even if they were unable to induce migration of DCs after binding their ligand(s). Spleen iDCs did not exhibit any Ca++ flux in response to CCL4, CCL5, CCL19, CCL20, or CCL21, although they did release Ca++ in response to a nonspecific Ca++ flux inducer, ionomycin (Fig. 3D and data not shown). iBMDCs, however, which migrated in response to CCL5, also fluxed after exposure of the DC to CCL5 (Fig. 3D) . As expected, mDCs fluxed in response to CCL19 and CCL21 but not to CCL4, CCL5, or CCL20 (data not shown).

CD8{alpha}+ and CD8{alpha}- iDCs do not localize to the spleen after i.v. injection
We have reported previously that allogeneic, liver-derived CD8{alpha}+ mDCs migrate to the DLN of recipients within 24 h of s.c. injection [31 ]. However, two other groups have been unable to detect fluorochrome-labeled spleen CD8{alpha}+ iDCs administered s.c. to syngeneic recipients in DLN [12 , 29 ]. Differences in protocols between studies, coupled with the present findings that at least both subsets of mDCs apparently express the intracellular CCRs necessary for migration, led us to investigate further the disparity in the reports regarding the ability of CD8{alpha}+ DCs to traffic in vivo. We first examined the patterns of migration of iDCs to the spleen after i.v. injection. The i.v. route was chosen, as it is the common route of DC administration for experimental antirejection or autoimmune disease therapy [4 , 57 , 58 ]. However, surprisingly, neither CD8{alpha}+ nor CD8{alpha}- splenic iDCs could be detected in the spleen of recipients 24 h after i.v. injection (Fig. 4 ), despite the reported ability of these DCs to prime naïve T cell responses via this route of administration [12 13 14 , 28 , 29 ]. To determine whether the pattern of splenic iDC traffic was related to their origin, iBMDCs were injected i.v. as controls. We were unable to detect iBMDCs in the spleen after 24 h, but these cells could be detected in the liver and lung 24, 48, and 72 h after injection (data not shown). These results were confirmed by rare-event flow cytometric analysis (see Fig. 6A ).



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  		Figure 4. mDCs but not iDCs are detected in T cell areas of the spleen 24 h after i.v. injection. Immunomagnetic bead-sorted CMFDA-labeled (green) CD8{alpha}+ or CD8{alpha}- B10 iDCs or mDCs (3–5x106) were injected i.v. into BALB/c mice. Twenty-four hours later, spleen sections were stained with biotinylated mAb anti-CD3 (T cell) and -CD19 (B cell), F4/80 (red pulp), MOMA-1 (marginal zone), and anti-CD11c (DC) followed by Streptavidin Cy3 (red). Few CMFDA-labeled CD8{alpha}+ or CD8{alpha}- iDCs localized to splenic T cell areas after i.v. injection. By contrast, mDCs of either subset were numerous in T cell and DC areas. 4',6-Diamidino-2-phenylindole (DAPI; blue) indicates nuclear staining. Original magnification, x200.

 


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  		Figure 6. i.v. injection is a more efficient route of administration for directing DC migration to secondary lymphoid tissues than s.c. injection. Flow-sorted, CMFDA-labeled DCs (3–5x106) were injected i.v. or s.c., and spleens or DLN were excised 24 later. Spleen–DC-enriched suspensions (A, C) and LN cell suspensions (B, D) were analyzed by flow cytometry for CMFDA-positive cells. Only CD8{alpha}+ and CD8{alpha}- mDCs were detected in spleens after i.v. injection, and the greatest number of DCs was at 24 h of the 72-h (data not shown) follow-up period. Although fewer CD8{alpha}+ mDCs were visualized by immunohistochemistry at 24 h, these DCs were apparent by flow cytometric analysis and in similar numbers to those for CD8{alpha}- mDCs. The data are from a single experiment representative of at least three performed. Numbers indicate the percentage of CMFDA-positive cells.

 
CD8{alpha}+ and CD8{alpha}- iDCs do not localize in DLN after s.c. injection
Despite data obtained in the rhesus macaque with blood iDCs that suggest otherwise [26 ], iDCs injected s.c. into the hind footpad of allogeneic recipient mice did not localize to the DLN 24 h after injection (Fig. 5 ), nor were the iDCs detected after rare-event flow cytometric analysis (Fig. 6B ) or in the footpad, as assessed by two-photon confocal microscopy (data not shown).



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  		Figure 5. mDCs colocalize with recipient DCs and T cells 24 h after s.c. injection. Immunomagnetic, bead-sorted, CMFDA-labeled (green) CD8{alpha}+ or CD8{alpha}- B10 iDCs or mDCs (3–5x106) were injected s.c. into one hind footpad of BALB/c mice. Twenty-four hours later, DLN sections were stained with biotinylated mAb anti-CD3 (T cell) and -CD19 (B cell), F4/80 (red pulp), MOMA-1 (marginal zone), and anti-CD11c (DC) followed by Streptavidin Cy3 (red). Few CD8{alpha}- iDCs localized to T cell areas after s.c. injection; no CD8{alpha}+ iDCs were visualized. Although either subset of mDCs was detected in T cell and DC areas at 24 h after s.c. injection, CD8{alpha}+ mDCs were found in far fewer numbers than their CD8{alpha}- counterparts. Blue, DAPI indicates nuclear staining; red, CD3 (T cell); CD19 (B cell); F4/80 (macrophage); CD11c (DC). Original magnification, x200.

 
CD8{alpha}+ and CD8{alpha}- mDCs colocalize with splenic T cells after i.v. injection
Unlike iDCs, both mDC subsets migrated to T cell areas (CD3+) and colocalized with (recipient) DCs (CD11c+) at 24, 48, and 72 h after i.v. injection (Fig. 4 and data not shown). Administered mDCs were not found in B cell follicles (CD19+), red pulp (F4/80+), or marginal zone (MOMA-1+). In keeping with the quantitative, rare-event data, fewer DCs were visualized over time (data not shown). The comparatively inferior CD8{alpha}+-to-CD8{alpha}- DC migration seen in the in vitro chemotaxis studies, however, was not borne out in the in vivo experiments, as both DC subsets were detected in similar numbers in the spleen at all time points (Fig. 6C and data not shown).

CD8{alpha}+ and CD8{alpha}- mDCs do not localize with DLN T cells after s.c. injection
Unlike previous reports [12 , 29 ], we detected CMFDA-labeled CD8{alpha}+ and CD8{alpha}- mDCs in the popliteal DLN [no DCs were detected in inguinal LN at any time point tested (data not shown)] 24 h after s.c. footpad injection (Fig. 5) . It is interesting that immunohistochemical analysis of the DLN revealed DCs dispersed throughout the tissue and not localized in T cell areas, as seen in the spleen. Nor did the DCs colocalize with B cells (CD19+) or macrophages [F4/80+; MOMA-1+ cells were not found in the LN (data not shown)]. CD8{alpha}+ DCs were furthered identified at 24 h by their faint costaining for PE–residual from flow sorting on CD8{alpha} expression immediately before injection (Fig. 6D) . Only CD8{alpha}- DCs were detected in DLN at 48 h, and neither subset was detected 72 h after s.c. injection (data not shown). Unlike rhesus macaque blood-derived mDCs [26 ], neither murine mDC subset was detected in the injected footpad mice as assessed by two-photon confocal microscopy (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have compared the migratory ability of murine CD8{alpha}+ spleen DCs to their better-studied CD8{alpha}- counterparts. Previous data regarding "classic" myeloid DCs, generated from BM or blood precursors or isolated from the skin or LN, suggest that iDCs are capable of responding to diverse, inflammatory CC chemokines. This is not surprising, given the role iDCs play during inflammation and in immune responses. However, unlike in vitro-generated BM- or blood-derived DCs, here, we have shown that iDCs isolated directly from spleen are more restricted in their chemotactic reactivity. Although iBMDCs exhibited positive (albeit weak, compared with mDCs) migration to iDC migration-inducing CC chemokines, as previously reported [18 , 22 , 23 , 36 , 37 , 39 , 40 , 53 ], spleen CD8{alpha}+ and CD8{alpha}- iDCs failed to respond to any of the CC chemokines tested. Preliminary studies in our laboratory with FL-mobilized murine liver (A. H. Lau, B. L. Colvin, and A. W. Thomson, unpublished observations) and kidney (P. Toby Coates et al., submitted) DCs reveal this phenomenon is not restricted to spleen iDCs, as iDCs isolated directly from these nonlymphoid/parenchymal organs also appear to have restricted chemotactic ability to CC chemokines. We find it unlikely that this phenomenon is related to DC mobilization by FL treatment, as FLDCs have been reported by several groups to be phenotypically and functionally identical to DCs from nontreated animals [4 , 14 , 48 , 59 ]. Studies using iBMDCs from FL-treated mice showed iBMDC responded to the same CC chemokines as those reported by groups using non-FL-mobilized, murine BMDCs [18 ], and our own data, using spleen iDCs from non-FL-treated mice, were comparable with that from FL-treated DCs (Fig. 2A) . Further, our in vivo migration data for freshly isolated, FL-mobilized DCs are consistent with that reported by Ruedl and Bachmann [12 ], who used untreated iDCs (Fig. 4) , and with that reported by Drake et al. [28 ], who used FL-treated mDCs.

It seems likely that spleen iDCs do not express functional CR, as evidenced by their lack of Ca++ flux (Fig. 3D) and lack of migration to their ligands (Fig. 2A) . At least one study has reported CCR5 expression on murine CD8{alpha}+ iDCs [through binding of FITC-labeled CCL4 (MIP-1ß); ref. 60 ], although we did not observe spleen CD8{alpha}+ iDC migration to CCR5 ligands (e.g., CCL4 or CCL5; Fig. 2A ). A possibility for the lack of detected activity is that the CCRs on iDCs have been down-regulated by constitutive expression of CC chemokines in the spleen. By RPA, we have detected low levels of CCL3 and CCL4 and high levels of CCL5 in the spleens of FL-treated mice (P. Toby Coates et al., submitted). Mack et al. [61 ] have shown that these three chemokines down-modulate CCR5 expression on murine natural killer cells in vitro (as well as CCR2 by CCL1 on monocytes) in a dose-dependent manner, suggesting that CCR expression down-modulation on iDCs in situ is a distinct possibility. No studies to date have reported CR expression on murine DCs using polyAb or mAb. Despite the availability of several such reagents, we were not able to demonstrate differential expression of CCRs for mouse DC subsets, as has been seen for mouse Th1 and Th2 cells [37 ] and human DCs [62 ].

We report here that spleen DCs up-regulate CCR6 mRNA expression after overnight culture (and subsequent phenotypic and functional maturation [4 ]), although neither iDCs nor mDCs responded to CCL20 (the natural ligand of CCR6). Iwasaki and Kelsall [30 ], conversely, reported that CD11b+(CD8{alpha}-) freshly isolated PPDCs migrated in response to CCL20, and neither CD8{alpha}+ PPDCs nor either subset of spleen DCs did so. Despite the fact that the freshly isolated DCs in their experiments appeared to be phenotypically immature, all the DC subsets evaluated migrated to CCL19 and CCL21, inconsistent with the present findings and those of others (Fig. 2A and 2B , and refs. [18 19 20 , 22 , 23 , 38 , 47 , 50 , 53 ]). Kucharzik et al. [63 ] detected enhanced green fluorescent protein/CCR6 on CD11b+(CD8{alpha}-) PP and spleen freshly isolated (untreated or FL-mobilized) DCs, although they did not attempt in either case to determine whether the receptor was functional or whether its expression changed with maturation. Varona et al. [64 ] also describe a lack of CD11b+ PPDCs in CCR6-/- mice, although they did not investigate whether this population was also absent in the spleen. Further, in the Iwasaki and Kelsall studies [30 ], DCs were isolated from spleens of BALB/c mice, and our DCs were isolated from the spleens of B10 mice. It has been reported that DCs from C57BL6/J mice express mRNA for Toll-like receptor (TLR)9 only, and BALB/c mice express mRNA for TLR2, -4, -5, and -6 [65 ]. Perhaps CR mRNA expression is strain- as well as tissue-of-origin-dependent. If indeed CCR6 is expressed on mouse spleen iDCs or mDCs, it appears to be nonfunctional and unable to be activated (as assessed by Ca++ flux and migration assays; Table 1 , Fig. 3 , and data not shown).

The lack of in vitro migration of splenic iDCs to CCL19 and CCL21 and their low expression of CCR7 mRNA are consistent with what has been reported for in vitro-generated iDCs. This unresponsiveness to constitutive lymphoid CC chemokines is further supported by the lack of retention of iDCs by the spleen after i.v. injection, presumably because of the inability of these DCs to respond to CCR7/ligands. This fact may also explain the absence of iDCs in DLN 24 h after s.c. injection. The lack of functional CCR7 on the cell surface precludes directed migration to secondary lymphoid tissue. iDCs become dispersed throughout the lung and liver after i.v. injection. It is not clear whether these circulating iDCs are capable of entering secondary lymphoid tissues after their activation by foreign Ag/inflammatory signals or if they are fated to recirculate until they die. Splenic mDCs, conversely, exhibit the migratory patterns of other mDCs reported thus far. In the present study, up-regulation of CCR7 mRNA expression in mDCs correlated with the ability to respond to CCL19 and CCL21 in vitro. This responsiveness was further borne out by not only the retention of mDCs in the spleen (up to 72 h after injection; data not shown) but by their mobilization to T cell areas. Comparatively fewer mDCs reached the T cell areas of DLN after s.c. infusion, and their habitation appeared more transient, with only CD8{alpha}- DCs persisting up to 48 h.

In the mature state, CD8{alpha}+ and to a greater extent CD8{alpha}- DCs responded to CCL19 and CCL21 in vitro. This disparity in the extent of migration to CCL19/CCL21 did not correlate with the level of in vivo traffic to the spleen and is not an index of a cell’s ability to mobilize to secondary lymphoid tissue. In vivo migration requires interaction with adhesion molecules and extravasation through lymphatic or blood vessel endothelia. Comparison of the two DC subsets clearly underlines the superior ability of CD8{alpha}- mDCs to traffic in vivo, as strikingly fewer CD8{alpha}+ than CD8{alpha}- mDCs were apparent in the DLN at all time points tested. We investigated whether the inferior ability of CD8{alpha}+ mDCs to migrate in vitro and in vivo compared with CD8{alpha}- mDCs correlated with surface expression of CCR7. Conceivably, lower expression of CCR7 would impair the ability of CD8{alpha}+ mDCs to migrate from peripheral to secondary lymphoid tissues. Aside from CCR7, two other DC markers, CD11b/CD18 (Mac-1) and CD205 (DEC-205), are worthy of consideration. CD11b/CD18 is an integrin that binds CD54, an adhesion molecule constitutively expressed on ECs [66 , 67 ]. The low-to-negative expression of CD11b/CD18 on CD8{alpha}+ DCs may have adverse effects on the ability of these DCs to interact with ECs for initiation of extravasation into (or out of) lymphatic vessels. CD205, a comparatively large glycoprotein expressed highly on CD8{alpha}+ DCs, might interfere with adhesion molecule interactions between CD8{alpha}+ DCs and ECs. Ongoing studies in our laboratory will attempt to determine what role, if any, either of these two molecules plays in the depressed ability of CD8{alpha}+ mDCs to migrate.

Our studies compare for the first time the in vitro and in vivo migration of CD8{alpha}- and CD8{alpha}+ DCs, taking into consideration the effect of the state of DC maturation on migratory capacity. In the earliest investigations of murine CD8{alpha}+ DC migration, freshly isolated spleen CD8{alpha}+ iDCs [12 , 29 ] were not detected in the DLN 24 h after s.c. injection, leading the authors to conclude that CD8{alpha}+ DCs could not actively migrate. Conversely, a few CD8{alpha}+ DCs were detected in the spleen after i.v. injection by rare-event flow cytometric analysis [12 ]. Here, we show that iDCs, mainly CD8{alpha}-, traverse to the DLN in barely detectable numbers. Even when CD8{alpha}- iDCs do reach secondary lymphoid tissues, they do not appear to be retained. Somewhat consistent with our results, Drake et al. [28 ] detected CD8{alpha}+ mDCs (alongside coincidently injected CD8{alpha}- DCs) in the DLN 24 h after s.c. injection. It was not determined, however, whether these CD8{alpha}+ DCs were identical to these injected or a subpopulation of CD8{alpha}- DCs that up-regulated CD8{alpha}+ upon CD40/CD154 engagement with DLN resident T cells, as has been reported for lymphoid chemokines [68 ]. The present study shows, for the first time, that CD8{alpha}+ iDCs do not migrate to secondary lymphoid tissues after s.c. or i.v. injection, whereas CD8{alpha}+ mDCs do, albeit less extensively after s.c. injection.

Here, we show the first Ca++ flux assays performed to determine CR activation in response to specific chemokines with in vitro-generated (iBMDC) and tissue-isolated (spleen iDC and mDC) mouse DCs. All other studies previous to this one have been undertaken with human DCs [62 , 69 ] or a murine iDC cell line [47 ]. Taken together with the migration data, these findings strongly suggest that DCs directly isolated from secondary lymphoid tissues (in particular, the spleen) do not respond to (inflammatory) CC chemokines. It is not clear from these studies whether the DCs do not express the receptors or if they have been desensitized. The eventual advent of reliable Ab for detection of mouse extracellular CR will aid these investigations greatly.

These studies additionally underscore the importance of the route of administration and the stage of DC maturation for potential therapeutic application. Although both DC subsets studied have been reported to be equally capable of T cell priming after s.c. or i.v. injection [12 13 14 , 28 , 29 ], it is probable that CD8{alpha}+ iDCs would have difficulty priming T cell responses after i.d. or i.p. injection, where a lack of resident migration-capable CD8{alpha}- DCs may preclude their cross-priming ability. CD8{alpha}- mDCs would appear a better choice in these instances. Our findings also suggest that efforts to maintain exogenous DCs in an immature state in vivo (to promote tolerance induction) may require a means to keep costimulatory molecule expression low (e.g., mAb blockade of CD40L [70 ]) but CCR7 expression high (e.g., retrovirally transduced CCR7 expression [71 ]) to aid in DC migration toward and retention within secondary lymphoid tissues. This knowledge will aid the design of strategies for selective targeting of DC subsets and as a consequence, manipulation of allo- and other immune responses [4 , 16 , 58 ].


   ACKNOWLEDGEMENTS
 
National Institutes of Health Grants RO1 AI41011 and RO1 DK49745 (to A. W. T.) and R21 HL69725 and R21 AI55027 (to A. E. M.) supported the authors’ work. The authors thank Dana Faratian for contribution to the early phase of this work. We also thank Alan Zahorchak for expert RPA techniques, Glenn Papworth and Simon Watkins for help with two-photon confocal microscopy, and P. Toby Coates, F. Jason Duncan, Amanda Schell, An de Creus, Masanori Abe, Zhiliang Wang, and Mark Hall for valuable discussion. Drs. Jean-Marc Navenot and Stephen C. Peiper (Medical College of Georgia, Augusta) provided invaluable advice on implementation of Ca++ flux assays.

Received December 18, 2002; revised October 2, 2003; accepted October 4, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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