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CD43 Deficiency Has No Impact in Competitive In Vivo Assays of Neutrophil or Activated T Cell Recruitment Efficiency¹

Biomedical Research Centre and Department of Pathology and Laboratory Medicine, University of British Columbia, British Columbia, Canada

	Abstract

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Using noncompetitive methodologies comparing CD43^+/+ and CD43^–/– mice, it has been reported that CD43^–/– leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43^–/– T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43^+/+ vs CD43^–/– activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43^+/+ and congenic CD43^–/– mice in either B6 or P-selectin^–/– recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

	Introduction

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During the inflammatory response, various cell types, including T cells and neutrophils, exit the vasculature and accumulate within inflamed tissue in a process collectively referred to as recruitment (1). Recruitment generally starts with rolling behavior of cells expressing selectin ligands along venule endothelia that express P- and E-selectin homing receptors (1, 2). Both E- and P-selectins are induced on inflamed endothelium in most tissues (3, 4), but constitutively expressed on skin endothelium in mice (5). The primary P-selectin glycoprotein ligand 1 (PSGL-1)³ is only functional after appropriate (including core 2 GlcNAcT-I (C2)) modifications of O-linked carbohydrates (6, 7) that are constitutively present in myeloid cells (8) and inducible in T cells (9, 10). E-selectin ligands (E-SelL), some requiring similar O-glycan modifications, are thought to include PSGL-1 (11, 12), but additional E-SelL, such as ESL-1, CD66, CD44, -2-3-sialyl Lewis x glycosphingolipids, and ₂ integrin, may also be active. Whether any of these alternate E-SelL contribute to physiologically relevant E-SelL functions is not clear.

Recent data based on in vitro observations of murine (13) and human (14) T cells have identified the activation-associated glycoform of CD43 as a potential novel ligand for E-selectin. These observations suggest that under some conditions, CD43 might facilitate rolling interactions and function as a homing receptor during E-selectin-mediated recruitment. The proposal that CD43 might function as an E-SelL was particularly surprising given previous work using intravital microscopy (IVM) describing a higher, not lower, frequency of rolling neutrophils and monocytes on activated venules in mice lacking CD43; notably, CD43^–/– leukocytes also exhibited retarded transmigration that collectively resulted in a net >2-fold reduction in recruitment (15). In terms of net consequences on recruitment, these observations found some support in reports in which in vivo T cell homing to secondary lymphoid organs (16) and recruitment to inflammatory sites (17) were inhibited with an Ab to CD43. In models of lymphocytic choriomeningitis virus (LCMV) infection and experimental autoimmune encephalomyelitis induction, CD43^–/– CD8 T cells (18) and CD4 T cells (19), respectively, exhibited reduced CNS recruitment.

In an effort to directly assess the influence of CD43 during recruitment, we have developed in vivo competitive methods to compare recruitment efficiencies of two or more cell populations at an inflammatory site within a single mouse. Such assays help eliminate the influence of environmentally rooted differences in the mouse strains being compared and indirect effects of a null mutation, transgene, or treatment on the organism that might affect recruitment (1). Using available fluorescent tracking dyes to mark competing cell populations, competitive in vivo recruitment assays have single-cell resolution, and thus the potential for sensitive quantitative analysis, even when recruited cell numbers are small.

We applied these competitive in vivo recruitment methods to assess efficiency of selectin ligand-dependent recruitment of control CD43⁺ vs CD43^–/– activated T cells to cutaneous inflammation and of peripheral blood neutrophils (PBN) to inflamed peritoneum. Lack of CD43 expression had no detectable impact on recruitment of activated T cells to inflamed skin, even in the absence of P-selectin, conditions in which recruitment was expected to be heavily E-selectin dependent. Similarly, C2-dependent recruitment of CD43-bearing and CD43-deficient PBN to inflamed peritoneum occurred with equal efficiency. Despite confirmation that the activation-associated glycoform of CD43 on activated T cells can perform as an E-SelL in vitro, CD43 deficiency had no detectable influence on recruitment in the two in vivo competitive recruitment models described. Our results suggest that the abundance of CD43-associated E-SelL on activated T cells is too low to be functionally relevant for recruitment.

	Materials and Methods

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Mice

Mice aged 8–20 wk were used for analyses. C57BL/6 (B6) mice were bred at the Biomedical Research Centre from founders obtained originally from The Jackson Laboratory. CD43^–/– mice (20) were bred at the Biomedical Research Centre from founders obtained originally from J. Green (Washington University School of Medicine, St. Louis, MO). These mice had been backcrossed on the B6 background for six generations by A. Sperling (University of Chicago, Chicago, IL), and were backcrossed on B6 for two more generations by us before intercrossing heterozygotes to yield CD43^–/– mice used (21). F7 B6 backcrossed C2^–/– mice (6) were provided by J. Marth (Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA). E-selectin^–/– mice (22) were provided by D. Bullard (University of Alabama, Birmingham, AL) and had been backcrossed on B6 mice in excess of 20 generations. PSGL-1^–/– and P-selectin^–/– mice on the B6 background were obtained from The Jackson Laboratory. PSGL-1^–/–CD43^–/– mice were bred in house from the stock strains noted above. The procedures used in this study were reviewed and approved by the University of British Columbia Animal Care Committee.

Medium and salt solutions

Cell suspensions were prepared in RPMI 1640 (11875-135; Invitrogen Life Technologies) supplemented with 8% FCS, 5 x 10^–5 M 2-ME, 100 U/ml penicillin, 100 U/ml streptomycin (StemCell Technologies), and 2 mM glutamine (Sigma-Aldrich). HBSS lacking Mg²⁺ and Ca²⁺ (Hanks^–) (catalog no. 14185-052; Invitrogen Life Technologies) was used in fluorescent tracking dye-labeling procedures. HBSS containing Mg²⁺ and Ca²⁺ (Hanks⁺) (catalog no. H8264; Sigma-Aldrich) was used as indicated.

Fluorescent dye labeling

CD8⁺ Con A blasts at 10⁷/ml were labeled with either 2 µM CFSE (catalog no. C-1157; Molecular Probes; Invitrogen Life Technologies) for 5 min at room temperature in Hanks^– or 10 µM CTO (cell tracker orange), and (5-(and-6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (catalog no. C2927; Molecular Probes; Invitrogen Life Technologies) for 10 min at 37°C in Hanks^–. After labeling, cells were diluted with an equal volume of culture medium, pelleted, washed once in Hanks⁺, and injected in Hanks⁺. For peripheral blood leukocyte labeling, cells were incubated with 0.1, 0.03, or 0.01 mmol CFSE in Hanks^– for 5 min at room temperature. An equal volume of rolling buffer (RB; see below) was then added, and cells were pelleted, washed once with Hanks^–, and injected in Hanks^–.

Activated T cells and the cutaneous inflammatory model

Activated T cells were generated by Con A stimulation. Con A primary stimulations were conducted under conditions that were previously described (23), with several minor modifications to most efficiently promote uniformly high levels of P-selectin ligand typically observed in peripheral CD8 T cell responses in vivo (23). Briefly, 10 ml of splenocyte cultures were prepared at 2 x 10⁶/ml with 4 µg/ml Con A (catalog no. 17-0450-01; Pharmacia; Pfizer) in 6-well Falcon 3046 plates (BD Biosciences). After a 2-day culture at 37°C in 5% CO₂, cells were harvested, washed, counted, and replated in secondary cultures at 0.25 x 10⁶/ml. After another 2 days, cells were harvested and refed for 1 more day. IL-2 used in these studies was included throughout the entire 5-day culture by supplementing them with 2.5% conditioned medium from murine IL-2 cDNA transfectants of myeloma X-653 provided by F. Melchers (Max Planck Institute for Infection Biology, Berlin, Germany). After the 5 days, 90–95% of cells in culture were CD8 T cell blasts.

For the cutaneous inflammatory model, mice were briefly anesthetized with isoflurane, their back skin was shaved, and 3 x 10 µl intradermal injections of Con A solution were administered (10 µg, filter sterilized, 1 mg/ml Con A in PBS). Between 5 and 10 million cells of each dye-labeled Con A blast population were then injected i.v. in a 300-µl volume. Eighteen to 20 h later, mice were either sacrificed by CO₂ and back skin was harvested or given a lethal i.p. dose of ketamine/xylazine and perfused as follows. Mice were perfused through the left ventricle using a peristaltic pump delivering at 7 ml/min with 20 ml of Hanks⁺, 20 ml of 2 µM CFSE in Hanks^–, and/or 12 ml of india ink solution. India ink solution was prepared with 2 parts commercial india ink (Pro Art black india ink, PRO-4100; DEMCO), 7 parts water, and 1 part 10x concentrated Hanks^–. Note that some other commercial india ink preparations produced prohibitively high red autofluorescence signals. After perfusion, incisions were made around the torso just below the arms and just above the legs. A midline incision from sternum to lower abdomen allowed the torso skin to be gently removed in a single sheet, rinsed in cold saline, mounted backside up on a rubber stopper, and secured in position under light tension with needles placed horizontally into the side of the stopper. This arrangement permitted relatively easy removal of subdermal muscle and connective tissue with fine forceps and a scalpel that was critical for visualization of s.c. vessels containing the infiltrates of interest. The stopper was immersed in short wide-mouthed beaker containing enough cold saline to just cover the mounted skin. Infiltrates were then imaged with a Leica MZ FLIII dissecting microscope outfitted with a x0.8–10 zoom objective and with blue (470 nm excitation; 515 nm barrier) and green (546 nm excitation; 590 nm barrier) filters to detect CFSE and CTO fluorescence, respectively. Images were captured with a microscope-mounted digital CoolSnap camera and imaging software (Photometrics) and overlayed on a computer with Adobe Photoshop. Cell counting was performed with public domain ImageJ software (http://rsb.info.nih.gov/ij/) using the Cell Counter plugin.

PBN and the peritoneal inflammatory model

PBN were prepared at room temperature and under conditions as free as possible from LPS contamination, according to a protocol provided on a website maintained by J. Lowe’s laboratory (University of Michigan, Ann Arbor, MI) (www.pathology.med.umich.edu/lowelab/mpmn.html). This protocol was designed to maintain normal neutrophil-rolling behavior on immobilized selectins. Some minor modifications were made. Briefly, blood was collected by cardiac puncture after administering CO₂ until respiration ceased. The chest cavity was opened and blood was collected from the right ventricle using a 22-gauge needle attached to a 3-ml syringe containing 100 µl of citrate phosphate dextrose anticoagulant solution. Blood was added to a 50-ml conical polypropylene tube (Falcon 2070) containing 25 ml of PBS/EDTA solution (2 mg/ml EDTA in PBS) at room temperature. One 50-ml tube was used for two or three mice depending on the amount of blood. Blood and PBS/EDTA were mixed, and tubes were spun at 1300 rpm for 10 min at room temperature. The supernatant was discarded by aspiration, and the loosened pellet was resuspended in 9 ml of LPS-free nanopure water for 30 s at room temperature to lyse most RBC. One milliliter of 10x PBS was then added to make the solution isotonic. The suspension was then diluted with 40 ml of RB containing 0.2% BSA, 10 mM HEPES, in Hanks^–. Tubes were then spun at 1300 rpm for 10 min at room temperature, and the supernatant was discarded. Cells were resuspended in 14 ml of RB and transferred to a 15-ml conical tube (Falcon 2095). Tubes were spun at 1300 rpm for 8 min at room temperature, and the supernatant was discarded. Cells were finally resuspended in 1 ml of RB, and an aliquot was labeled with Gr-1 for neutrophil counting. The remaining PBL were labeled with either CFSE or CTO, as described above. For counting, 100 µl of 10-µm latex beads at 10⁶/ml (2–1000; Interfacial Dynamics; Invitrogen Life Technologies) was mixed with an equal volume aliquot of PBL labeled with AlexaFluor 488-conjugated Gr-1 (prepared in house). The bead/cell mixtures were subjected to flow analysis in the presence of 500 ng/ml propidium iodide to determine the ratio of propidium iodide^lowGr-1^high PBL:beads. This ratio was directly proportional to PBN count. Fluorescent tracking dye-labeled PBL were injected i.v. into mice that had just received an i.p. injection of either autoclaved thioglycolate (3% w/v dissolved in water; Difco, 243010), or 2% oyster glycogen (G8751; Sigma-Aldrich) dissolved in water (24). Recipient mice received approximately one mouse equivalent of purified donor PBL. Three to four hours later, peritoneal exudates were harvested, stained with allophycocyanin-conjugated Gr-1 (17-5931-82; eBioscience), and analyzed for Gr-1^highCFSE⁺ events.

Western blotting and E-selectin-human Ig (hIg) analysis

Activated T cells were prepared, as described above, and lysed for immunoprecipitation of either E-SelL or CD43. Cell lysis and E-selectin-hIg immunoprecipitation were performed, as previously described (13), with some modifications. Briefly, cells were washed thrice in PBS and lysed at 3 x 10⁷/ml in 1% Triton X-100, 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM CaCl₂, and 1 mM PMSF for 30 min at 4°C. Three hundred microliters of lysate was preabsorbed with 50 µl of protein A-Sepharose CL-4B (17-0780-01; Pharmacia) for 1 h at 4°C and then for 90 min with either 10 µg of hIgG (I4506; Sigma-Aldrich), 10 µg of H18, or 10 µg of E-selectin-hIgG chimera (575-ES; R&D Systems) immobilized on 20 µl of protein A-Sepharose CL-4B. H18 is a peptide affinity-purified rabbit Ab specific for the cytoplasmic domain of CD43 recognizing all glycoforms of CD43 (25). As a further specificity control for E-selectin-hIg precipitation, 25 mmol EDTA was included during the immunoprecipitation with E-selectin-hIg, where indicated. Protein A-Sepharose was then washed thrice with lysis buffer, and the immunoprecipitate was eluted with 25 µl of gel-loading buffer consisting of 4% lauryl sulfate, 125 mM Tris (pH 6.8), 0.1 mg/ml bromphenol blue, and 20% glycerol. H18 immunoprecipitates were reduced with 5% 2-ME, whereas E-SelL immunoprecipitates were not; both immunoprecipitates were then heated to 90°C for 5 min and loaded (2 µl of the H18 immunoprecipitate or the entire E-selectin-hIg immunoprecipitate) onto a 7% polyacrylamide gel for electrophoresis and electroblotting onto Hybond ECL nitrocellulose (Amersham Biosciences). After transfer, blots were blocked with 5% skim milk powder in TBS (pH 8.0) for 30 min and then probed for 60 min with TBS containing 1 µg/ml H18, 0.5% Tween 20 (BP337-500; Fisher Scientific), and 0.5% skim milk powder in TBS. Blots were then washed with TBS-Tween (TBS with 0.5% Tween 20) five times, 5 min per wash, and then probed with HRP-conjugated anti-rabbit Ig in TBS-Tween containing 0.5% skim milk powder for 60 min. Blots were then washed, as described above, twice with TBS alone, and then H18 bound to immunoprecipitated and immobilized CD43 was finally detected with ECL reagent (Amersham Biosciences) and autoradiography with BioMax film (Kodak).

E-SelL on activated T cells was detected by flow cytometry with the same E-selectin-hIg reagent described above and anti-hIg PE (109-116-098; Jackson ImmunoResearch Laboratories). To identify small differences in E-SelL-hIg binding to PSGL-1^–/– vs PSGL-1^–/–CD43^–/– cells, B6, PSGL-1^–/–, and PSGL-1^–/–CD43^–/– cells were stimulated, as described above, except that they were seeded at 0.75, 1.0, and 1.5 x 10⁶/ml for the initial Con A stimulation and then maintained independently for the next 5 days. E-selectin-hIg staining and analysis were performed in triplicate on all cultures. Cells were costained with, and gated on, cells binding CD8 FITC (553031; BD Pharmingen). E-selectin-hIg staining of 30,000 CD8⁺ events was recorded for each sample. Geometric means of E-selectin-hIg staining were determined for all triplicates from cultures at all three densities and all three mouse strains using CellQuest software (BD Biosciences) and compared using two-tailed Student’s t test based on sample sets with equal variance (Excel software; Microsoft).

	Results

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Development of competitive in vivo recruitment model of cutaneous inflammation

Our investigations began with development of a skin recruitment model whereby fluorescent dye-labeled activated T cells expressing high levels of selectin ligand could be injected into a mouse and subsequently visualized at an inflammatory site using a fluorescence dissecting microscope. Efficient recruitment of T cells is currently thought to rely on TCR engagement that acts in concert with chemokine signals to promote firm adhesion and trigger transmigration (26, 27). s.c. Con A administration is reported to stimulate a relatively efficient, P-selectin-, and E-selectin-dependent, s.c. accumulation of T cells (28, 29). Indeed, data implicated a more pronounced role for E-selectin in this recruitment model (29). We therefore chose to test the activity of Con A as an inflammatory agent in vivo and its capacity to attract in vitro activated T cells labeled with fluorescent tracking dyes.

Fluorescently labeled cells were injected i.v. into mice immediately after they had received s.c. injection of 10 µl (10 µg) boluses of Con A. At this dose, a nonulcerating cutaneous inflammation was induced that peaked at day 1 and largely subsided by day 2. The s.c. inflammatory site was examined for accumulations of CFSE- or CTO-labeled dye-positive cells. Preliminary studies showed that dye positive cell accumulations were clearly evident at 18–20 h and followed vascular tracts in the highly vascular region between the s.c. muscle layer and the base of hair follicles; at later time points, infiltrating cells were more abundant, but also more diffusely distributed. Using this experimental procedure, we then assessed whether these cell accumulations reflected a physiologically relevant recruitment process and evaluated the feasibility of competitive recruitment studies in single mice.

As shown in Fig. 1A, when two genetically identical donor cell populations were labeled with different fluorescent tracking dyes and injected into Con A-treated recipients, accumulations of both cell types were clearly present and at apparently equal frequency. To determine whether the use of Con A as an inciting agent altered the requirements typically involved in physiological recruitment, the efficiency of activated C2^–/– T cells was compared with activated B6-derived T cells; C2^–/– T cells lack the ability to generate the branched O-glycan structure on selectin ligands, especially PSGL-1 (6, 30), that are recognized by selectins and necessary for activated T cell recruitment (31). As shown in Fig. 1B, C2^–/– cells accessed s.c. Con A inflammatory sites very poorly relative to B6 T cells. This observation provided evidence that the conditions for accumulation of activated donor T cells relied on effective selectin ligand formation, and thereby replicated physiologically relevant recruitment processes. When CD43^–/–-activated T cells were compared with B6 T cells, as shown in Fig. 1C, there was no obvious difference in accumulation efficiency. This result was somewhat unexpected, as recruitment of CD43^–/– neutrophils to inflamed peritoneum had been reported to be defective (15). These results prompted us to quantify the infiltrates and develop a competitive peritoneal recruitment assay for neutrophils.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. s.c. Con A supports C2-dependent accumulation of activated T cells. Con A-activated T cells from day 5 cultures from B6, CD43–/–, or C2–/– mice were labeled with CFSE or CTO and injected i.v. into B6 recipient mice that had just previously received three 10 µl s.c. Con A injections. Eighteen hours later, mice were sacrificed and back skin was prepared for imaging. Three representative x90 magnification images are shown: A, B6 (CTO, red) + B6 (CFSE, green); B, B6 (CTO, red) + C2–/– (CFSE, green); C, B6 (CTO, red) + 43–/– (CFSE, green).

To verify that the accumulation of activated T cells at s.c. sites of Con A-induced inflammation reflected a de facto recruitment process, we first needed to demonstrate that donor lymphocytes actually exited the vasculature, did so through postcapillary venules in which lymphocyte emigration occurs most readily (32, 33), and where P (34)- and E-selectins (35) are most abundantly expressed. Vasculature exit was important to establish because transmigration was reportedly impaired in leukocytes lacking CD43 (15). To this end, we developed a simple staining method to discriminate between arteries and veins. We had previously developed a method of i.v. perfusion with CFSE to assess vascular parameters in an Alzheimer disease mouse model (36). In further development of this technique, we found that CFSE perfusion preferentially labels the arterial vascular tree, as shown in Fig. 2, A and B, but labeling is virtually absent on the venous side. This preferential labeling of the arterial vasculature does not reflect biochemical differences between arterial and venous endothelium (as evident in tail vein perfusion studies and high CFSE concentration cardiac perfusion studies; D. Carlow, data not shown), but appears to reflect rapid absorption of CFSE label out of circulation due to the large endothelial cell surface area encountered in the capillary bed. By perfusing with CFSE and then india ink, arterial and venous vascular trees could be readily visualized, as shown in Fig. 2C. When this imaging method was applied to the Con A inflammation model, as exemplified in Fig. 2D, it was evident that cells extravasated, and did so as expected through venules, not arterioles.

View larger version (101K): [in this window] [in a new window]   FIGURE 2. Activated T cells extravasate at the anatomically correct position in the Con A-induced cutaneous inflammation model. To demonstrate arterial staining with CFSE, terminally anesthetized mice were perfused with CFSE. Back skin was prepared for imaging of the arterial vascular tree: A, Original magnification, x8; B, original magnification, x100 (A inset blow up); C, original magnification, x8; mice were additionally perfused with india ink to highlight the venous tree; D, CFSE and india ink perfusions were performed on a mouse that had received, 18 h previously, s.c. injection of Con A and i.v. injection of tracking dye-labeled Con A blasts: B6-CTO (red) and B6-CFSE (green).

To establish the Con A inflammation procedure as a valid recruitment model, it was essential to show that activated C2^–/– T cells persisted normally in vivo despite their failure to accumulate s.c. (Fig. 1B). Thus, in vitro viability, in vivo recovery from spleen, and s.c. accumulation of activated donor T cells were assessed in mice that had received B6 and CD43^–/– cells or B6 and C2^–/– T cells. As shown in Fig. 3, A and B, CD43^+/+, CD43^–/–, and C2^–/– cell viability were maintained in vitro and in vivo (spleen) after tracking dye labeling. When imaging was performed on skin from these mice, infiltrates were counted. Four independent images of infiltrates per mouse were analyzed. Raw count data for red and green cells are summarized in Fig. 3C. C2^–/– cells exhibited a clear disadvantage in accessing the s.c. Con A site, whereas CD43^–/– T cells and CD43^+/+ B6 T cells accessed the Con A site comparably. The data shown in Fig. 3C were further compiled into ratios of green:red cells for each image. Ratios generated for each of the four images from a single mouse were averaged, and the corresponding SD was calculated, as shown in Fig. 3D. In summary, despite the persistence of viable activated C2^–/– T cells in vivo, they accessed the Con A-induced s.c. inflammation poorly, confirming that selectin ligand formation was required and validating this recruitment model. Furthermore, CD43^–/– T cells exhibited a frequency of recruitment that was not significantly different from CD43^+/+ T cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. C2–/– T cell blasts are maintained in vivo, but fail to recruit in the Con A cutaneous inflammation model, whereas CD43–/– blasts are recruited normally. Four mice received s.c. Con A injections together with tracking dye-labeled i.v. injection of mixtures of B6 and CD43–/– Con A blasts (mixtures 1 and 2) or B6 and C2–/– Con A blasts (mixtures 3 and 4). Dyes were reversed in different mixtures (1 vs 2 and 3 vs 4) to control for dye-related trafficking artifacts. An aliquot of each cell mixture used for injection was placed in culture with IL-2 to monitor dye toxicity. After 18 h, the relative survival of each dye-labeled population was assessed by flow cytometry of in vitro cultured cells (A), by flow cytometry of recipient spleen cell suspensions (B), and by ImageJ software-assisted counting of red vs green cell signals in fluorescence images of the cutaneous inflammation site (four images per mouse; C). The cell-counting data shown in C were compiled as average ratios of CFSE:CTO cell scores determined and shown in D. Recipient mice 1–4 received the following cell-dye combinations: mouse 1 received B6-CFSE plus CD43–/– CTO; mouse 2 received CD43–/– (CFSE) plus B6 (CTO); mouse 3 received B6 (CFSE) plus C2–/– (CTO); and mouse 4 received C2–/– (CFSE) plus B6 (CTO). The data shown are representative of five independent experiments.

To assess neutrophil recruitment to inflamed peritoneum, we applied competitive recruitment methods using PBN. Three modifications of the competitive recruitment methodology developed for T cells described above were introduced. First, thioglycolate and oyster glycogen were used as inflammatory stimuli, the latter being the agent formerly used in experiments describing the defect in CD43^–/– neutrophil recruitment. Second, to reduce the possibility of dye artifacts affecting recruitment, CFSE alone was used as a tracking dye. Of the fluorescent tracking dyes available, CFSE is relatively intense and is tolerated well. Three low concentrations of CFSE were used that allowed simultaneous flow cytometric enumeration of three independent PBN populations. Third, PBN from peripheral blood were used instead of in vitro activated T cells. Preliminary analysis indicated that despite their low numbers in peripheral blood, native PBN were superior to LPS-elicited or G-CSF-elicited PBN for peritoneal recruitment. PBN can be effectively distinguished by their level of the cell surface marker Gr-1 (37), and this reagent was used to follow PBN specifically. The concentration of viable neutrophils in sample PBL preparations from each mouse strain was determined. PBL were then labeled with distinct CFSE concentrations, combined with other PBL preparations to yield a mixture of PBL containing equivalent PBN numbers from each strain, as shown in Fig. 4A, and then injected i.v. into mice that had just received either thioglycolate or oyster glycogen i.p. Three hours later, PBL and peritoneal exudate cells from the recipient mice were harvested, labeled with Gr-1-APC, and analyzed by flow cytometry for Gr-1^high CFSE-labeled cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Normal recruitment of peripheral blood-derived CD43–/– neutrophils to inflamed peritoneum. PBL from B6, C2–/–, and CD43–/– mice were isolated. Viable Gr-1high PBN were identified and counted, as described in Materials and Methods. PBL from B6, C2–/–, and CD43–/– mice were then labeled with 0.1, 0.03, or 0.01 µm CFSE, respectively, and mixed in proportions that, on the basis of the count data, yielded an input ratio of 1:1:1 ratio of B6, C2–/–, and CD43–/– Gr-1high neutrophils. An aliquot of this input mixture was stained with Alexafluor 488-conjugated Gr-1– and propidium iodide to confirm equivalent input numbers of viable Gr-1high cells from each mouse PBL preparation. As shown in A, gated analysis of the Gr-1high input mixture demonstrated essentially equivalent numbers of the three CFSE-labeled subpopulations. B6 recipient mice received an i.p. injection of either thioglycolate (TG) or oyster glycogen (OG) and then an i.v. injection of the 1:1:1 Gr-1high mixtures of B6, C2–/–, and CD43–/– neutrophils. Three hours later, PBL (B) and peritoneal exudate cells (PEC) (C) were isolated, stained with Gr-1-APC, and analyzed for abundance of each dye-labeled population. Using the recovery of B6 cells () as a reference, defined as 1 in each experiment, the ratio of recovered C2–/– (shaded) and CD43–/– () relative to B6 was compiled for each of four experiments, as shown in D. Each triplet histogram summarizes CFSE+ cell yields from one mouse. OG1/PBL refers to analysis of peripheral blood harvested from a mouse 1 h after i.p. injection of oyster glycogen; OG2/PBL corresponds to blood analysis 2 h after injection, etc. IN, Corresponds to input ratios of cells at time of injection for each experiment. Data from four experiments are shown in the four separate clusters of histograms.

Assessment of E-selectin and CD43-dependent recruitment using in vivo competition

Recent in vitro studies to elucidate E-SelL identified the activation-associated glycoform of CD43 as a potential target. Interestingly, CD43 appears to serve selectively as an in vitro ligand for E-selectin, failing to bind P-selectin-Ig chimeras (13, 14). As a cell surface mucin, CD43 is normally highly substituted with O-glycans that can be further modified by C2 upon activation (25) and, at least in human cells, can develop the sialyl Lewis x structure used to generate selectin ligands (38). Our failure to detect a CD43-dependent effect on recruitment in the previous experiments could reflect a redundant function of P- and E-selectins. We therefore sought to analyze CD43-dependent effects on recruitment under conditions in which the influence of P-selectin was eliminated. P-selectin^–/– recipients were therefore used with the intent to eliminate recruitment supported by P-selectin and reveal recruitment activity dependent upon E-selectin. The Con A-induced in vivo recruitment of activated T cells appeared to be well suited to address a potential role of CD43 in E-selectin-dependent recruitment because involvement of E-selectin in this system had been previously established (28, 29). To address whether CD43 can contribute to recruitment activity, activated T cells from B6, C2^–/–, CD43^–/–, and PSGL-1^–/– mice were generated and paired against B6 control cells in competitive in vivo recruitment assays to cutaneous inflammation in B6, P-selectin^–/–, or E-selectin^–/– recipients.

Three to four images from each of the recipients shown in Fig. 5 were scored for control B6 and competitor dye-positive cells. These raw count data were expressed as a ratio of competitor:control for each image, and these ratios compiled with ratios from two additional experiments, as shown in Fig. 6. The image data exemplified in Fig. 5 illustrated several points. First, recruitment of C2^–/– (Fig. 5B) and PSGL-1^–/– (Fig. 5D) donor cells was severely compromised, as expected; recruitment of CD43^+/+ or CD43^–/– T cells in B6 recipients (Fig. 5, A and C) was comparable, confirming observations presented in Figs. 1 and 3. Second, overall recruitment of CD43^+/+ donor (green) cells in P-selectin^–/– and in E-selectin^–/– recipients (Fig. 5, E–H) was reduced relative to B6 recipients (Fig. 5, A–D), reinforcing the view that both selectins were present and contributing to recruitment in this model. Third, in P-selectin^–/– recipients, efficient recruitment maintained dependence on both C2 and PSGL-1 in that cells lacking these genes recruited poorly relative to B6 cells, as shown in Fig. 5E (C2^–/–, red; B6 donor cells, green) and Fig. 5G (PSGL-1^–/–, red; B6 donor cells, green). This result was consistent with the activity of PSGL-1 as a known E-SelL. Finally, B6 CD43^+/+- and CD43^–/–-activated T cells were recruited comparably in P-selectin^–/– recipients, indicating that in this in vivo T cell recruitment model, lack of CD43 did not affect recruitment efficiency in P-selectin^–/– recipients in which recruitment should depend heavily on E-selectin. In summary, despite compelling data demonstrating that CD43 can bind to CD43 (13, 14), lack of CD43 had no detectable impact on recruitment efficiency in the Con A skin recruitment model described above.

View larger version (100K): [in this window] [in a new window]   FIGURE 5. CD43 does not facilitate E-selectin-dependent recruitment of activated T cells. Competitive recruitment of CFSE-labeled control B6 Con A-activated T cell blasts and CTO-labeled blasts from either B6, C2–/–, CD43–/–, or PSGL-1–/– (as indicated in each panel) was coinjected into B6 recipients (A–D), P-selectin–/– recipients (E–G), or an E-selectin–/– recipient (H). All recipients had received s.c. injections of Con A, as described in Materials and Methods, at the time of cell injections. Eighteen hours later, mice were perfused with india ink and skin was prepared for imaging. Representative images are shown from one of four independent, and qualitatively consistent, experiments.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Compilation of image analysis data. Three to five images from each recipient shown in Fig. 5, together with similar image analysis data from two additional experiments, were compiled. In all cases, competitive recruitment was performed with syngeneic activated B6 control cells vs competitor (X) B6, CD43–/–, C2–/–, or PSGL-1–/– cells in either B6, P-selectin–/–, or E-selectin–/– recipients. The competitor/recipient combinations are indicated on the y-axis. Cells labeled with CFSE vs CTO were enumerated as in Fig. 4, and the ratios of competing cell:B6 control were determined for each image, averaged for all images analyzed from a given recipient, and plotted. The averaged ratios with corresponding SD of competitor:B6 cell recruitment represented an efficiency of recruitment relative to B6.

We anticipated that if CD43 contributed significant E-SelL function in vivo, it should do so under conditions in which the enzymes forming the selectin ligands on known substrates, such as PSGL-1, are active. Previous studies demonstrating E-SelL interaction with CD43 from cultured murine or human T cells used CD43 purified from anti-TCR-activated T cells that were maintained in IL-2. Furthermore, only the high m.w. (activation-associated) glycoform of CD43 exhibited E-selectin-binding activity (13, 14). The T cells we used for the cutaneous recruitment assay were also activated and maintained in IL-2, conditions in which both the activation-associated glycoform of CD43 and P-selectin ligands were heavily expressed (39). However, if activated T cells used in our analyses lacked, for whatever reason, E-SelL on CD43, then conclusions about CD43 function as an E-SelL during in vivo recruitment would not be possible. Therefore, it was necessary to confirm that CD43 molecules on the activated T cells used to assess in vivo recruitment in fact bore de facto E-SelL. To this end, lysates of activated T cells were immunoprecipitated with E-Sel-hIg, and the eluates were subjected to Western blotting with anti-CD43 Ab. As shown in Fig. 7, A and B, a high m.w. form of CD43 was effectively immunoprecipitated by E-selectin-hIg, but not by either hIgG or E-selectin-hIg in the presence of EDTA, confirming that CD43-E-SelL was indeed present in activated T cells used in the in vivo recruitment analyses. Use of activated T cells from PSGL-1^–/– vs PSGL-1^–/–CD43^–/– mice enabled direct resolution of the PSGL-1-dependent, CD43-dependent, and PSGL-1/CD43-independent E-SelL. Cell surface staining with E-selectin-hIg chimera shown in Fig. 7, C and D, demonstrates that the contribution of CD43-E-SelL was minor, but significant (p < 0.01) when compared with PSGL-1-dependent E-SelL and other nonPSGL-1/non-CD43 E-SelL. These data are consistent with the previous in vitro observations that CD43-E-SelL can be present on activated T cells (13, 14), and also consistent with our failure to observe CD43-E-SelL-dependent contributions during in vivo recruitment of the activated T cells we have used, presumably due to the low abundance of this entity.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. E-selectin binds CD43 on activated T cells. The presence of CD43-specific E-SelL on activated T cells used for the in vivo recruitment assays was confirmed by E-selectin-hIg immunoprecipitation (ip) of CD43 from lysates of such T cells. Rabbit anti-mouse CD43 polyclonal Ab H18 is CD43 cytoplasmic domain specific and was used to probe Western blots of anti-CD43 (H18) immunoprecipitates (A) or E-selectin-hIg chimera immunoprecipitates (B) of activated T cells, as described in Materials and Methods. A, First lane, H18-Sepharose control; second lane, H18-Sepharose ip of activated T cell lysate; third lane, Sepharose-alone control ip of activated T cell lysate. B, First lane, hIg-Sepharose ip of activated T cell lysate; second lane, E-selectin-hIg-Sepharose ip of activated T cell lysate; third lane, E-selectin-hIg-Sepharose ip of activated T cell lysate in the presence of EDTA. C, CD43-specific E-SelL resolved by E-selectin-hIg staining of activated CD8 T cells from B6 mice with (heavy solid line) or without (...) EDTA, from PSGL-1–/– mice (light solid line), and from PSGL-1–/–CD43–/– mice (...). D, Compiled geometric means of E-selectin-hIg staining of activated CD8+ T cells from B6, PSGL-1–/–, PSGL-1–/–CD43–/– mice () together with corresponding negative controls in which EDTA was included during staining (). Error bars shown correspond to 1 SD. Two-tailed Student’s t test indicated statistical significance of reduced E-selectin-hIg staining of PSGL-1–/–CD43–/– relative to PSGL-1–/– (p < 0.01). CD43-specific E-SelL-hIg binding was confirmed in six independent experiments.

	Discussion

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Con A was used to induce s.c. inflammation and has been shown to be a relatively effective recruiting agent in vivo (28). The efficacy of Con A in this regard may stem from its ability to engage the TCR directly (40), thereby providing the TCR signal thought to be necessary for efficient T cell recruitment (26, 27). Con A also appears to have innate immunity-stimulating activity, as it can induce expression of B7 costimulatory molecules (41, 42) and is also effective at short-term recruitment (4 h) of nonlymphoid cells in peripheral blood to a cutaneous site (D. Carlow, data not shown).

Although we have not assessed in vivo CD43-E-selectin interactions directly, our results do not support the proposition that CD43 can function as an E-SelL in vivo as in vitro observations would suggest (13, 14). Because rolling is generally a prerequisite for subsequent adhesion and recruitment, it was assumed that if CD43 offered a substantive rolling function via E-selectin, it would translate into improved recruitment particularly in P-selectin^–/– recipients. Despite the lack of substantial CD43-dependent activated T cell cutaneous recruitment, CD43-E-SelL could be demonstrated both biochemically and by cell surface staining with E-selectin-hIg chimera on these T cells (Fig. 7). FACS analysis revealed that CD43-E-SelL was in low abundance relative to E-SelL on PSGL-1 and other ill-defined (non-PSGL-1, non-CD43) substrates. The low abundance of CD43-E-SelL is consistent with our inability to find in vivo relevance for CD43-E-selectin interactions. Notably, preliminary analysis of E-SelL-hIg staining of PSGL-1^–/– vs PSGL-1^–/–CD43^–/– PBN revealed a similar, very minor, contribution of CD43-E-SelL (data not shown). It is quite possible that in a different model of inflammation, an in vivo contribution of CD43 as an E-SelL might be more apparent. However, we have noted both our evidence (re: Fig. 5) and other evidence (28, 29) that E-selectin participates significantly in the Con A-induced cutaneous recruitment model described. Whether alternative T cell activation could promote a more significant expression, and in vivo function, of CD43-E-SelL remains to be determined and is currently under investigation in our laboratory.

Woodman et al. (15) used IVM to document a >2-fold inhibition in peritoneal recruitment by leukocytes, predominantly neutrophils, in CD43^–/– mice relative to CD43^+/+ control mice. This outcome was thought to reflect the net consequence of increased adherence to intravascular endothelium and inhibited transmigration by CD43^–/– leukocytes. The techniques we have applied do not resolve differences in rolling or transmigration efficiency, but our data demonstrate normal net recruitment efficiency among CD43^–/– cells in the assays described. Possible explanations for these conflicting results include the use of noncompetitive experimental formats or CD43^–/– mice that were not formally congenic. Onami et al. (18) recently reported that CD8 T cells in CD43^–/– mice recruited poorly to LCMV-infected CNS compared with CD8 T cells in CD43⁺ control mice, and suggested that the CD43^–/– recruitment defect was restricted to the CNS sites of inflammation. It would be interesting to test whether the observed difference in recruitment efficiency would persist in a competitive recruitment model of LCMV-specific B6 vs B6.CD43^–/– congenic T cells to LCMV-infected CNS.

Several further aspects of the in vivo recruitment models developed in this report deserve highlighting. First and foremost, the assays described are relatively simple and accessible; the fluorescence dissecting microscope was the only technical equipment required. Multiple dye-labeled tracking methods have been used extensively for lymphocyte trafficking studies in secondary lymphoid organs, but much less frequently in recruitment analyses. Cutaneous IVM imaging through ear skin (43) has been successfully used in analysis of selectin-selectin ligand interactions in noninflamed contexts (5, 44), but inflammatory agent access through ear skin is limited and visualization through inflamed ear skin is difficult (45). The recruitment assays described in this work lack the real-time advantages of IVM that have proven so useful in documenting intravascular events in recruitment, but they circumvent mechanical and technical challenges in visualizing exteriorized tissue associated with IVM (45). When compared with tissue exteriorization in IVM techniques, the techniques we describe are noninvasive. These methods provide a static snapshot, capturing the outcome of recruitment events, but doing so in a relatively simple, rapid, quantifiable, internally controlled procedure with multiparameter capacity. These assays should help to fill a noted gap (1) in experimental methods by providing one option for relatively clear and direct visualization of in vivo T cell recruitment to extralymphoid tissues in a competitive format.

Regarding the neutrophil recruitment assay, methods were applied to isolate neutrophil-rich nucleated cell suspensions from peripheral blood. With minimal manipulations required, including CFSE labeling and adoptive transfer, apparently normal recruitment behavior was preserved in notoriously labile Gr-1^high neutrophils. This approach made competitive in vivo recruitment analyses feasible and the conclusion that absence of CD43 did not alter recruitment efficiency. With clear evidence of C2 dependence and absence of evidence for CD43-dependent neutrophil recruitment, the question of whether CD43 provides an E-SelL on neutrophils would seem moot.

In summary, we have developed and presented two experimental methods to assess recruitment efficiency of activated, CD43-deficient, T cells to inflamed skin, and of neutrophils to inflamed peritoneum in a competitive in vivo format. The use of competitive recruitment methods provides leverage in resolving authentic variation in recruitment efficiency. These models were used to evaluate whether loss of CD43 significantly affected recruitment; experiments in both models indicated that loss of CD43 had no impact.

	Acknowledgments

 
We acknowledge Dr. Maki Ujiie for stimulating discussions during development and validation of the cutaneous in vivo recruitment model, and for helpful suggestions after proofreading the manuscript.

	Disclosures

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The authors have no financial conflict 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 a grant from the Canadian Institutes for Health Research (Grant MOP-64267).

² Address correspondence and reprint requests to Dr. Douglas A. Carlow, Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia, Canada V6T-1Z3; E-mail address: doug{at}brc.ubc.ca or Dr. Hermann J. Ziltener, Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia, Canada V6T-1Z3; E-mail address: Hermann{at}brc.ubc.ca

³ Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand 1; C2, core 2 GlcNAcT-I; CTO, cell tracker orange; E-SelL, E-selectin ligand; hIg, human Ig; IVM, intravital microscopy; LCMV, lymphocytic choriomeningitis virus; PBN, peripheral blood neutrophil; RB, rolling buffer.

Received for publication May 12, 2006. Accepted for publication August 15, 2006.

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