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* Department of Immunology and Microbiology,
Department of Orthopedic Surgery, and
Department of Internal Medicine, Section of Rheumatology, Rush University Medical Center, Chicago, IL 60612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Sustained migration of leukocytes from the blood into the synovial tissue and cavity of multiple joints is a hallmark of the chronic inflammatory process in both PGIA and RA. Cells of both the adaptive and innate immune system are recruited to the joints; T and B cells are more frequent in the synovial tissue than in the joint cavity, whereas polymorphonuclear granulocytes (neutrophils) constitute the major cell population in the synovial fluid (2, 3, 5, 9, 10, 11). To gain entry into a target tissue, leukocytes implement a sequence of adhesive interactions with the vascular endothelium, collectively known as the "multistep paradigm" of extravasation (12). Leukocytes first transiently tether to, then roll on, the endothelium under blood flow. Next, chemokine-triggered activation of leukocyte integrins leads to firm adhesion of these cells to the endothelium so that they resist detachment from endothelial cells (13, 14). Finally, leukocytes migrate across the vessel wall into the tissue (12).
L-selectin (CD62L), the lymphocyte homing receptor (15), and CD44, the hyaluronan receptor (16), are two of the major adhesion molecules expressed by leukocytes of both lymphoid and myeloid origin (17). In addition to its established role in the homing of lymphocytes to lymph nodes (LNs), CD62L has been shown to mediate leukocyte rolling on inflamed endothelium (18, 19, 20). CD44 has been shown to function primarily by supporting leukocyte rolling at inflammatory sites (21, 22), but recent observations in CD44 gene knockout (KO) mice suggest that loss of CD44 might facilitate lymphocyte homing to peripheral LNs during inflammation (23, 24).
In a dermal delayed-type hypersensitivity model, it was shown that mice deficient in CD62L could not mount a response to epicutaneous immunization due to the inability of naive lymphocytes to home to the skin-draining LNs where they could acquire memory and effector functions (18, 25). Expression of CD62L was also required for the prompt initiation of leukocyte migration into the knee joint in Ag-induced arthritis, a nonautoimmune, monoarticular form of joint inflammation (26). In experimental allergic encephalomyelitis, transfer of wild-type (WT) macrophages to CD62L-deficient mice was necessary to cause myelin damage in the CNS (27). Previous work from our laboratory demonstrated that DBA/1 mice deficient in CD44 developed collagen-induced arthritis at reduced incidence and severity despite normal autoimmune responses to type II collagen (28). In contrast to CD62L KO lymphocytes that migrate poorly to LNs (18, 25), lymphocytes from CD44-deficient mice showed an increased propensity to home to LNs following immunization with type II collagen, bacterial superantigen (23), or injection with conalbumin (24). The role of CD62L in autoimmune arthritis has not been investigated. Because of the intricate regulatory pathways involved in the generation of autoimmunity to a joint (cartilage) component, it is expected that induction of a chronic autoimmune form of arthritis, such as PGIA, would be difficult in CD62L KO mice. We hypothesized that the LN homing defect (18) could be corrected, at least in part, by introducing CD44 deficiency into CD62L-null mice (i.e., creation of a double KO). "Restoration" of the adaptive immune system could then allow for induction of an autoimmune response to PG in CD44/CD62L double KO mice. Dual adhesion receptor deficiency is, on the other hand, expected to manifest in impaired leukocyte recruitment at the periphery (19, 21, 26), thus resulting in reduced inflammation and joint damage.
To dissect the requirement for CD44 and CD62L for the autoimmune vs the inflammatory response in PGIA, we generated double CD44/CD62L-deficient mice in the PGIA-susceptible BALB/c background and compared their Ag-specific T cell responses, autoantibodies titers, and arthritis development to those in WT, CD44 KO, and CD62L KO mice upon immunization with hPG. Using intravital video microscopy (IVM), we also compared the recruitment of leukocytes to the ankle joints in WT and single and double KO mice and examined the relative involvement of leukocyte subpopulations in joint inflammation. In addition, we tested the ability of single and double KO lymphocytes to migrate to the secondary lymphoid organs of SCID mice and to adoptively transfer PGIA. We found that inflammatory, but not autoimmune, responses were compromised by the lack of CD44 and CD62L and that expression of these molecules in granulocytes was required for the development of severe arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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WT BALB/c mice were purchased from the National Cancer Institute (NCI). Mice deficient in CD44 (in the DBA/1LacJ background) were generated by targeted gene disruption as described previously (28). CD62L-null mice (in the C57BL/6 background) (29) were purchased from The Jackson Laboratory. BALB/c mice lacking CD44 or CD62L were obtained by backcrossing into the BALB/c background, using a genomic marker-assisted speed congenic method (5, 30), where heterozygous KO males with the most complete BALB/c genetic makeup were mated with WT BALB/c females. Using this selection step before each backcross, full BALB/c background was attained faster (7–9 backcrosses) than with random cross-breeding (which usually requires >12 backcrosses). Full BALB/c background was confirmed by whole genome screening with 244 simple sequence-length polymorphic markers (30). Finally, CD44+/–, CD62L+/–, and CD44+/–/CD62L+/– heterozygous males and females of pure BALB/c background were intercrossed, and offspring, homozygous for single and double gene mutations, were identified by genomic PCR (26). The phenotype of adult mice (cell surface CD44 and CD62L expression or lack thereof) was also confirmed by flow cytometry. Female SCID mice of BALB/c background (NCI/NCrC.B-17-scid/scid), used for cell transfer experiments, were obtained from the NCI and were maintained under germfree conditions. All animal procedures were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Rush University Medical Center.
Induction of primary PGIA and adoptive transfer to SCID mice
hPG was isolated from osteoarthritic cartilage, which was obtained from patients undergoing joint replacement surgery, and was provided through the Orthopedic Tissue, Transplant, and Implant Repository of Rush University Medical Center, with the approval of the Institutional Review Board. PG was extracted from pooled cartilage samples as described in our previous publications (3, 4, 5, 6). WT, single KO, and double KO female BALB/c mice at 10–16 wk of age were immunized i.p. with 100 µg of hPG (measured as protein) in dimethyldioctadecyl ammonium bromide adjuvant four times at 3-wk intervals, according to an established protocol (5, 6). Adoptive transfer of PGIA to SCID mice was performed as described previously (31). In brief, each SCID mouse was injected i.p. with 5 x 107 spleen cells from donors with primary PGIA, along with 200 µg of hPG. Two weeks later, each mouse received 2.5 x 107 donor spleen cells and 100 µg of hPG. Animals were inspected for symptoms of arthritis after the third immunization and the first cell transfer for primary and adoptive PGIA, respectively. According to the presence of redness (erythema) and degree of swelling, the paws were visually scored on a scale of 0–4 per paw (0–16 per mouse). The severity of arthritis in each paw was determined as follows: 0, no swelling or redness; 1, mild swelling and erythema; 2, moderate swelling; 3, severe swelling; and 4, severe swelling with hardening of the periarticular tissue (5, 6). The same investigator inspected and scored the paws twice a week in a blinded manner.
Cell isolation, flow cytometry, and histology
Blood was collected in heparin-containing tubes from anesthetized mice through retro-orbital puncture. At sacrifice (wk 0, 8, or 11 of immunization), the spleen and joint-draining (axillary, brachial, inguinal, and popliteal) LNs were harvested, and single-cell suspensions were prepared. RBC in the spleen and blood samples were eliminated by hypotonic lysis. Cell counts and viability were determined by trypan blue exclusion. Ankle synovial fluid cells were collected after repeated flushing of the open joint cavity with PBS, followed by gentle scraping of the synovial intima with a pipet tip. Cells in LN, spleen, blood, or joint fluid samples were immunostained with fluorescence-conjugated mAbs against adhesion molecules, including CD44, CD62L, and P-selectin glycoprotein ligand 1 (PSGL-1), as well as to leukocyte subpopulation markers such as CD3, CD45R/B220, Gr-1 (granulocytes) (BD Biosciences), and F4/80 (monocytes/macrophages) (Serotec). Nonspecific binding of the fluorescence-conjugated mAbs to FcRs was inhibited by preincubation of the cells with unlabeled mAbs to CD16 and CD32 (Fc block; BD Biosciences). Flow cytometry was performed using a FACSCalibur instrument and CellQuestPro software (BD Biosciences). The hind limbs of naive or arthritic mice were dissected, fixed in formalin, decalcified, and embedded in paraffin. The tissue sections were stained with H&E.
Assays of Ag (PG)-specific immune responses
These assays were performed as described before (5, 6, 7, 31). In brief, spleen or LN cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (HyClone) in round-bottom 96-well plates (2 x 105 cells/200 µl/well, in triplicate wells) with or without hPG (25 µg protein/ml). To determine hPG-specific cell proliferation, the cells were pulsed with [3H]thymidine (1 µCi/well) on the fourth day of culture and harvested on day 5. Radioisotope incorporation was measured by scintillation counting and expressed in counts per minute. PG-specific IL-2 production by spleen cells was determined using a CTLL-2 bioassay, where IL-2 bioactivity was measured in the supernatants after 24 h of culture. To determine the PG-specific production of IFN-
and IL-4, spleen cells were cultured for 5 days using the same cell density and Ag dose as indicated for the proliferation assays. The concentration of IFN- and IL-4 in the culture supernatants was measured using ELISA (BD Biosciences).
PG-specific Abs in serum were measured by ELISA (6, 31). Briefly, mice were bled at defined time points, and serum samples were stored at –20°C. Maxisorp microplates (Nunc) were coated with hPG or mPG (0.5 µg protein/well in sodium carbonate coating buffer (pH 9.5)) overnight at 4°C. Fat-free milk was used to block nonspecific protein binding sites. Serially diluted serum samples and internal standard samples (pooled sera from arthritic mice with known anti-PG Ab titers) were incubated with the immobilized PGs, and plate-bound hPG- or mPG-specific Abs were detected using peroxidase-conjugated rabbit IgG against mouse IgG1 and IgG2a isotypes (Zymed Laboratories), respectively.
Intravital video microscopy
IVM on the mouse ankle synovium was performed as described previously (11). In brief, the mouse was anesthetized by i.m. injection of a xylazine-ketamine mixture. The ankle was immobilized on a custom-built stage, and the Achilles tendon was cut off the calcaneus to expose the ankle synovial tissue to the intravital fluorescence microscope (model Eclipse E600FN; Nikon). The synovium of the open ankle was continuously superfused with warm PBS through a fine silicon tube attached to the objective lens. To visualize leukocytes in the circulation, the mouse was injected i.v. with 10 µg of rhodamine 6G (Molecular Probes), a cell-permeable fluorescent DNA-binding dye (11, 26). Leukocyte trafficking was recorded by streamline acquisition of images with a digital video camera (CoolSnap CF; RS Photometrics) fitted to the microscope. Blood vessel diameters were measured, and leukocyte adhesion behavior was analyzed offline using Metavue software (Universal Imaging). Leukocytes present in postcapillary venules with diameters ranging from 20 to 50 µm were selected for analysis. The parameters measured were the number of rolling leukocytes per 103 µm (1 mm) vessel perimeter per minute, rolling velocity (micrometer per second), and the number of adherent leukocytes per 105 µm2 of endothelial surface per minute (11, 26). For in vivo detection of CD44 and CD62L expression or the presence of granulocytes in the synovial vessels, mice were injected with fluorescence-conjugated mAbs against CD44, CD62L, or Gr-1 (4 µg mAb/mouse) instead of rhodamine 6G.
Lymphocyte trafficking assays
To compare the ability of WT and KO spleen cells to migrate to lymphoid or extra-lymphoid organs, we performed short-term mixed-cell homing assays (23, 28). First, lymphoid tissue (and homeostasis) was reconstituted in the immunodeficient mice by i.p. injection of splenocytes (5 x 107 cells with 200 µg of hPG) from immunocompetent WT or KO BALB/c mice with primary PGIA, as described for the adoptive transfer experiments. Two weeks later, the reconstituted SCID mice received a mixture of WT and KO spleen cells from arthritic donor mice after labeling the donor cells with stable cell-tracing fluorophores of different colors. WT spleen cells were labeled with either CellTracker Green (catalog no. C7025; 3 µM) or CellTracker Orange (catalog no. C2927; 5 µM) and KO cells with CellTracker Orange, according to the manufacturer’s instructions (Molecular Probes). The green fluorescent (WT) and "orange" (red) fluorescent (WT or KO) spleen cells were mixed at a 1:1 ratio, and a total of 2 x 107 viable cells (with 100 µg of hPG) in a final volume of 150 µl was injected i.v. into each SCID recipient. SCID mice previously reconstituted with WT splenocytes received a mixture of red and green fluorescence-labeled WT donor cells, and those injected with KO splenocytes received a mixture of red KO and green WT donor cells (23). The hosts were killed 24 h later, and single-cell suspensions were prepared from peripheral blood, spleen, joint draining LNs, and ankle synovial fluid. Labeled donor cells in these suspensions, along with those in the original samples of mixed donor cells (before injection), were detected using two-color flow cytometry. The net ratios of red:green fluorescent donor cells that had migrated into SCID tissues were calculated after normalizing the actual ratios of red:green-labeled cells retrieved from the host’s LNs and spleen (RLN and RSpl, respectively) to the ratios of red:green donor cells in the mixtures before injection (RInj) (23). SCID mice injected with a 1:1 mixture of red and green fluorescent WT donor cells served as internal controls, where the migration efficiency of differentially labeled WT cells was expected to be identical.
Statistical analysis
Data were analyzed using one-way ANOVA (SPSS). The post hoc Dunnett’s t test was used to determine significant differences (p < 0.05) between the WT and any of the three KO groups of mice. For time course experiments conducted on the same sets of mice, repeated measures ANOVA was also performed to determine significant differences (p < 0.05) between the groups in the overall responses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To determine whether expression of CD44 and CD62L was required for the development of PGIA, we immunized four genotypes (WT, CD44 KO, CD62L KO, and double KO) of BALB/c mice with hPG. To achieve maximum PGIA incidence and severity in all groups, mice were given four i.p. injections of hPG in adjuvant at wk 0, 3, 6, and 9. They were monitored for the onset of PGIA and the severity of arthritic symptoms until wk 11. As shown in Fig. 1A, the incidence of PGIA was reduced in all KO mice as compared with age-matched WT animals. CD62L KO and double KO mice also showed a delay in arthritis onset, but the majority (70–100%) of the mice developed arthritis by wk 11 regardless of genotype (Fig. 1A). The severity of PGIA was reduced in both single and double KO mice relative to WT (Fig. 1B). This difference was significant for the CD62L KO and double KO groups until the end of the observation period (wk 11) and for the CD44 KO group on wk 7, 9, and 10 (Fig. 1B).
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Ag-specific cell proliferation is reduced, but cytokine and Ab responses are normal in hPG-immunized CD44KO, CD62L KO, and double KO mice
CD62L has been shown to play a crucial role in LN homing and subsequent activation of lymphocytes upon contact with Ag (18, 19, 20). Therefore, the delay in arthritis development and reduced disease severity, particularly in mice lacking CD62L (Fig. 1B), could be the consequence of inadequate response to PG immunization due to impaired lymphocyte homing. We compared the magnitude and kinetics of immune responses in the four genotypes of mice by measuring PG-specific LN and spleen cell proliferation, production of IL-2, IL-4, and IFN- by splenocytes in vitro, as well as serum levels of mPG-specific autoantibodies (both Th1- and Th2-polarized isotypes) at wk 0 (before immunization), wk 8 (2 wk after the third PG injection), and wk 11 (2 wk after the fourth PG immunization). The hPG-specific proliferation of spleen cells from all KO mice was significantly weaker than the proliferation of WT splenocytes at both wk 8 and 11 of immunization (Fig. 2A). LN cells from the immunized mice (including WT) generally gave very poor proliferative responses to in vitro stimulation with hPG; proliferation was essentially undetectable in all KO LN cultures at wk 8 but was detectable by wk 11 in CD44 KO LN cultures (Fig. 2A). IL-2 was produced in approximately equal amounts by hPG-stimulated splenocytes, with the highest levels in double KO cultures, but IL-2 could not be detected in the LN cell cultures (data not shown). PG-specific IFN- production by spleen cell cultures was comparable in all genotypes, and less IL-4 was found in the CD44KO and double KO than in the WT cultures at wk 8 but not at wk 11 (Fig. 2B). Serum levels of mPG-specific IgG2a and IgG1 autoantibodies were not reduced in KO mice; in fact, we found significantly elevated levels of the IgG2a isotype of these autoantibodies in serum samples of CD44 KO mice at both wk 8 and 11 and of the IgG1 isotype in the sera of CD62L mice at wk 11 of immunization (Fig. 2C). Taken together, except for a reduced proliferative response of spleen cells from all three groups of KO mice to in vitro Ag stimulation (which correlated well with the reduced severity of PGIA in these groups), adaptive immunity did not seem to be compromised in PG-immunized CD44 KO, CD62L KO, and double KO mice.
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As both CD44 and CD62L have been shown to support leukocyte rolling (18, 20, 32, 33, 34), the reduced severity of arthritis, observed in mice lacking one or both of these receptors (Fig. 1B), could also be attributed to impaired leukocyte rolling and subsequent recruitment in the target organs. We performed IVM on mouse ankle joints (11) to determine whether the adhesion behavior of leukocytes in the synovial postcapillary vessels of ankle synovium were different in the four genotypes of mice. Both nonarthritic and arthritic animals were used for IVM experiments. In the absence of CD44 or CD62L or both, we expected the number of leukocytes that rolled on synovial endothelium to decrease, and rolling velocity to rise, as a result of loss of one or two major receptors that can mediate rolling. However, the number of rolling cells was not reduced in the synovial vessels of arthritic CD44 KO and CD62L KO ankles and was even significantly elevated in double KO, compared with WT mice (Fig. 3A). The velocity of rolling cells was significantly higher in double KO than WT mice (Fig. 3B). A compensatory increase in the circulating pool of leukocytes expressing PSGL-1, another adhesion molecule that mediates rolling via endothelial or platelet selectin binding (35), could explain the increased propensity of double KO cells to roll, but we found little evidence for such compensation (the average proportion of PSGL-1+ cells was 31% in WT and 39% in double KO blood). Although the percentage of leukocytes expressing CD62L was lower in CD44 KO than in WT mice (11 and 22%, respectively), the absence of CD44, combined with low CD62L expression in CD44 KO cells, did not result in increased rolling interactions (Fig. 3A) or elevated rolling velocity (Fig. 3B). Importantly, the number of firm adherent cells was reduced in the vessels of all KO mice and was significantly lower in CD62L and double KO than in WT mice (Fig. 3C). The difference between WT and KO leukocytes, with respect to firm adherence to endothelium, remained consistent when WT and KO ankle joints with similar arthritis scores were compared (data not shown).
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PGIA can be adoptively transferred to syngeneic SCID mice using spleen cells isolated from BALB/c donors with primary PGIA (31). SCID mice lack functional T and B cells, but they possess an intact innate immune system. Therefore, lymphocyte transfer to SCID mice could provide help in determining whether expression of CD44 and CD62L in the innate or adaptive arm of the immune system is more important for development of a severe form of adoptive PGIA. If expression of CD44 and/or CD62L in the adaptive immune system is required for severe disease, then SCID mice reconstituted with splenocytes from CD44 KO, CD62L KO, or double KO donors with low-severity PGIA are expected to develop mild arthritis. However, SCID mice that received spleen cells from arthritic KO donors all developed PGIA promptly (Fig. 4A) and with similar or even higher severity (Fig. 4B) than their counterparts reconstituted with cells from arthritic WT donors. This observation suggested that the development of adoptive disease was not hampered by the reduced ability of KO donor spleen cells to proliferate in the presence of PG (Fig. 2A) and that host (SCID)-derived cells significantly contributed to disease severity. Indeed, in vivo immunostaining identified leukocytes expressing CD44 and CD62L not only in the inflamed synovial venules of SCID mice reconstituted with WT donor cells (Fig. 4C, left panel), but also in the vessels of SCID hosts that received double KO (CD44/CD62L-negative) splenocytes (Fig. 4C, right panel). This provided in vivo evidence for the recruitment of host (innate immune) cells at the site of inflammation in PGIA.
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As spleen (not LN) cells were used for the transfer of PGIA from arthritic donors to SCID mice, we could not rule out the possibility that CD62L-independent occupancy of the SCID LNs by the splenocytes accounted for the induction of severe disease, particularly in animals injected with cells from CD62L-deficient or double KO donors. Furthermore, spleens from arthritic donors could contain an "effector" population of either lymphoid or nonlymphoid cells, capable of migrating to the joints directly. To investigate these possibilities, we conducted in vivo trafficking assays by transferring fluorescence-labeled spleen cells from arthritic WT and KO donors into SCID mice. As described in Materials and Methods, WT donor splenocytes were labeled with a green fluorophore and mixed at 1:1 ratio with red fluorescence-labeled cells from each genotype of the KO donors, which allowed for simultaneous detection of WT and KO cells upon their retrieval from the SCID organs 24 h after transfer. As shown in Fig. 5, A and B, spleen cells from CD44 KO donors migrated significantly better than WT cells to the LNs of SCID hosts, whereas LN homing of CD62L KO cells was impaired severely. Because CD44 KO splenocytes demonstrated enhanced LN occupancy, it was expected that CD44 deficiency could compensate for this homing defect of CD62L-null cells. This was not the case, however, as splenocytes lacking both CD62L and CD44 showed very modest improvement in LN homing compared with spleen cells from CD62L single KO mice (Fig. 5, A and B). In contrast to LNs, the spleen was occupied with similar efficiency by WT and KO donor splenocytes, although relative to WT cells, the ratio of CD44 KO cells was slightly decreased, and the ratios of CD62L and double KO cells were increased in the spleens of SCID mice (Fig. 5, C and D). We were unable to detect fluorescent donor cells in the ankle synovial fluid of SCID mice 24 h (or even 72 h) after transfer (data not shown).
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As described above, severe PGIA developed promptly in SCID mice after transfer of KO spleen cells (Fig. 4B), although the LNs were poorly populated in mice reconstituted with cells from CD62L KO or double KO donors (Fig. 5), and arthritogenic donor splenocytes did not seem to migrate to the joint cavity. On the other hand, following transfer of unlabeled spleen cells from double KO donors to SCID mice, leukocytes expressing CD44 and CD62L (obviously of host origin) were detected by IVM in the synovial vessels of inflamed SCID joints (Fig. 4C). These observations indicated that innate immune cells significantly contribute to the severity of joint inflammation in PGIA. The most likely candidates are the granulocytes because these cells (mainly neutrophils) are present in large numbers in the joints of mice with PGIA (3, 5, 11) and other models of immune-mediated arthritis (26, 36), as well as in the inflamed joints of RA patients (37). Therefore, we investigated the relationship between granulocyte influx and disease severity, first focusing on PGIA in WT mice. IVM following in vivo immunostaining for Gr-1 revealed the presence of very few (mostly rolling) granulocytes in the synovial venules of noninflamed ankles (score: 0), but the number of cells that interacted with the endothelium increased in correlation with the progression of arthritis (from score 1 to 3) in these joints (Fig. 6A). The relative number of granulocytes in the joint fluid followed a similar pattern: very few Gr-1+ cells were detected by flow cytometry in the fluid of noninflamed ankles, but the proportion of granulocytes "skyrocketed" with arthritis progression (Fig. 6B). This trend was also observed on histological sections of the ankles: as inflammation advanced, more and more polymorphonuclear leukocytes accumulated in both the synovial tissue and the joint cavity of the affected ankles (Fig. 6C). When comparing the cellular composition of synovial fluid cells from WT and KO joints of matching inflammation scores, we found that the proportions (%) of Gr-1+ cells were somewhat lower in KO than WT joints at a low inflammation score but were similarly high (>90%) at an advanced degree of inflammation (Fig. 6D). T and B cells and monocytes/macrophages were minor constituents of the synovial fluid exudate in both WT and KO mice, and, in contrast to granulocytes, these mononuclear cell populations did not show much change in size during arthritis progression (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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As expected, the incidence and severity of arthritis were reduced in the mutant animals as compared with WT mice, and resistance to severe arthritis was more pronounced in mice lacking CD62L (single or double KO) than in those lacking CD44 only. These observations, which suggest that CD62L might play a greater role than CD44 in the development of an inflammatory joint disease, resonate with our previous study where mice lacking CD62L were found to be more resistant to a nonautoimmune form of arthritis than CD44 KO mice (26). It is conceivable that the generation of the autoimmune response upon active immunization (as in PGIA) requires a high level of coordination within the adaptive immune system by means of directed cell migration to, and acquisition of immunological memory in, the lymphoid compartments. Subsequent development of the inflammatory disease also requires cooperation between adaptive and innate immune system players at the periphery. The question arises as to which processes and players are affected the most by the loss of CD62L and/or CD44 expression in PGIA.
The crucial role of lymphocyte CD62L in LN homing has been established (15, 18). Although CD44 has not been directly implicated in this process, we (23) and others (24) found that CD44-deficient lymphocytes accumulated in LNs with greater efficiency than WT cells after in vivo activation. Based on these findings, it was expected that Ag (both hPG and mPG)-specific immune responses would be severely reduced in PG-immunized CD62L-deficient mice, enhanced in CD44 KO, and close to normal in CD44/CD62L double KO mice. Besides a surprisingly low magnitude of PG-specific LN cell proliferation (relative to spleen cells), we found that the absence of CD62L expression had a negative impact on the Ag-specific response of LN cells in PG-immunized mice. Lymphocytes from the spleens of all KO mice exhibited reduced proliferative responses to hPG, for which there is no clear explanation at this time. However, the same cells demonstrated normal cytokine production, suggesting that the signaling function of splenocytes was preserved in the KO mice. Compared with WT mice, autoantibodies against mPG of both IgG2a and IgG1 isotypes were produced with equal or greater efficiency in all KO mice, indicating that the lack of CD62L and CD44 expression had no major effect on autoantibodies production (4, 5) or the Th1/Th2 balance associated with PGIA (7).
How could CD62L KO mice mount a globally "normal" (auto)immune response in the face of a LN homing defect? The answer most likely lies in the systemic (i.p.) route of Ag administration used to induce PGIA. As confirmed by our cell transfer results in the present study, CD62L is required for LN homing, but it is not necessary for lymphocyte entry into the spleen (40). The requirement for CD62L for acquisition of full immunological memory was therefore bypassed by the i.p. route of immunization, allowing for Ag delivery to the spleen, which lymphocytes could enter in a CD62L-independent fashion. The weak LN (vs strong spleen) immune responses in the PG-injected mice suggest a greater contribution of the spleen than of the LNs to the generation of immunological memory, but the best evidence for the prominent role of the spleen in PGIA development is the ability of splenocytes from arthritic BALB/c donors to transfer disease to immunodeficient mice (8, 31). In the present study, spleen cells from WT, CD62L KO, CD44 KO, and double KO BALB/c mice were equally prompt and effective in inducing severe arthritis upon transfer to SCID mice. This suggested that in PGIA, the spleen was capable of gathering lymphocytes and APCs in a CD62L- and CD44-independent manner, as well as providing a tissue microenvironment for the assembly and function of the adaptive (auto)immune system. Furthermore, the cell transfer studies indicated that splenocytes from PG-immunized CD62L KO and double KO donors that readily populated the spleen, but migrated poorly to the LNs of SCID mice, were still potent in "setting the stage" for severe joint inflammation in the SCID host. This raised the possibility that the failure of these arthritogenic spleen cells to trigger severe disease in the primary form of PGIA could be due to inefficient recruitment of KO effector cells to the inflammatory site.
Using IVM, we found that leukocytes in the synovial venules of double KO mice with primary PGIA exhibited significantly increased rolling interactions, elevated rolling speed, and a reduced ability to adhere firmly to endothelium as compared with leukocytes in WT mice. The simplest explanation for the increased proportion of rolling cells in double KO mice is that leukocytes, which interacted with the endothelium but failed to adhere, kept rolling. In this respect, the lack of CD62L and CD44 function could have been compensated for by PSGL-1 and perhaps also by "partially activated" integrins that are able to support rolling (41). It is likely that double KO cells were more easily carried away in a fast rolling motion by the shear force of the blood flow than their better-equipped WT or single KO counterparts. Fast rolling, in turn, shortened the exposure of leukocytes to chemokines, thus lowering the rate of integrin-mediated arrest and firm adhesion (14). In the synovial microcirculation of inflamed joints, all KO leukocytes demonstrated decreases in firm adhesion compared with WT cells in the order of double KO < CD62L KO < CD44 KO < WT, which correlated quite well with the severity of primary PGIA in these groups of mice. As firm adhesion is a committed step that is usually followed by transendothelial migration (12), decreased leukocyte adherence could lead to reduced extravasation and less severe inflammation. As noted above, spleen cells from these mildly arthritic KO donors were able to trigger severe arthritis in SCID mice, suggesting that the bulk of leukocytes migrating into the joints in primary PGIA did not represent the cells of the adaptive immune system.
We showed that leukocytes expressing CD44 and CD62L were being recruited in the synovial venules of SCID recipients after transfer of splenocytes from double KO donors, thus providing in vivo evidence for the participation of host-derived innate immune cells in the local inflammatory process. Furthermore, in primary PGIA, we demonstrated a positive relationship between the degree of inflammation and the abundance of granulocytes (neutrophils) that interacted with synovial endothelium and subsequently migrated into the synovial tissue and joint cavity. Although T and B cells, as well as macrophages, could be identified in both noninflamed and inflamed joints, these cells were present in low proportions, and their numbers did not increase markedly with arthritis progression. Collectively, these results suggest that in both the primary and adoptive forms of PGIA, the severity of inflammation largely depends on the abundance of innate immune cells (granulocytes) in the joint. We also found that the ankle joint fluid of KO mice contained fewer granulocytes than the synovial fluid of WT mice at a low degree of inflammation in primary PGIA, but KO granulocytes "caught up" with WT cells in ankle joints at an advanced stage of inflammation. These results, together with the IVM data, suggest that the lack of CD62L and CD44 expression lowers the rate of granulocyte influx into the joints, thereby delaying the onset and slowing down the progression of PGIA. However, as we have shown earlier in WT mice with PGIA (11), at an advanced stage of inflammation (score 
3), the joints become saturated with inflammatory cells, leading to a decline in the rate of leukocyte extravasation. Thus, with slowly continuing recruitment, KO granulocytes could catch up and eventually infiltrate the joints to the same extent as WT cells did. This was supported by the observation that the severity of primary PGIA, although reduced initially in CD44 KO, CD62L KO, and double KO mice, reached a degree similar to WT by wk 15 of immunization (B. Sarraj and K. Mikecz, unpublished data). Adoptively transferred PGIA developed and progressed in a WT-like fashion in SCID mice reconstituted with KO spleen cells, suggesting that CD44 and CD62L, expressed in the hosts’ innate immune system, facilitated leukocyte recruitment. Therefore, the innate immune cells that contributed mostly to disease severity must have been neutrophil granulocytes from the SCID mice. Using IVM after in vivo coadministration of fluorescent mAbs to Gr-1 and CD44, we found that the majority of leukocytes interacting with the synovial endothelium of SCID mice (which developed arthritis upon transfer of unlabeled double KO spleen cells) coexpressed Gr-1 and CD44 (B. Sarraj and K. Mikecz, unpublished results), indicating that most of the cells recruited in the inflamed joints were indeed host-derived granulocytes.
Murine neutrophils lacking CD62L (42) or CD44 (43) have been shown to exhibit impaired recruitment in vivo in response to, for example, local stimulation with proinflammatory cytokines such as TNF-
or chemokines. The requirement for CD62L (20) or CD44 (22) for T cell migration into inflammatory sites has been proposed but not firmly established. Because of the paucity of T cells that extravasate in the joint in PGIA (11), it is difficult to ascertain whether expression of these adhesion receptors is necessary for the migration of T lymphocytes to this site. Our adoptive transfer study suggests that, if CD62L and/or CD44 had been critical for the entry of activated (arthritogenic) T cells into the joint, inflammation would have been reduced in SCID mice reconstituted with lymphocytes lacking one or both of these adhesion molecules, but this was not the case.
Taken together, neutrophil granulocytes emerge as the major inflammatory cell population reliant on the expression of CD62L and, to a lesser extent, of CD44 for efficient influx into the joint in PGIA. In RA, neutrophils emigrate from postcapillary venules located in the synovial sublining area into the joint cavity in quite large numbers, causing severe damage to the cartilage matrix (9, 37, 44). Among other factors, elevated serum concentrations of TNF-, and the presence of autoantibodies-containing complement-fixing immune complexes in the joint, have been implicated in neutrophil recruitment in RA (39). As demonstrated by in vivo studies, TNF--activated vascular endothelium promotes neutrophil recruitment (45), and autoantibodies-containing immune complexes (e.g., serum from arthritic K/BxN mice) preferentially localize to the joints and initiate neutrophil-dependent arthritis upon transfer to naive mice (36, 46). These finding suggest that TNF- and autoantibodies, which are mainly (TNF-) or exclusively (autoantibodies) produced by adaptive immune cells, can access the joint and attract granulocytes, thereby effectively connecting the adaptive and innate arms of the immune system in susceptible individuals. Therefore, the clinical efficacy of TNF-- and B cell-targeting therapies in RA can be, in part, attributed to inhibition of neutrophil recruitment by reducing the interactions between adaptive and innate immunity (47, 48). Agents that antagonize CD62L and CD44 function, downstream of these interactions, could provide further aid in the therapeutic effort to suspend neutrophil influx into the joints in RA.
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1 This work was supported in part by National Institutes of Health Grants RA45652 and RA051163. 
2 Address correspondence and reprint requests to Dr. Katalin Mikecz, Department of Orthopedic Surgery, Rush University Medical Center, Cohn Research Building, Room 712, 1735 West Harrison Street, Chicago, IL 60612. E-mail address: Katalin_Mikecz{at}rsh.net
3 Abbreviations used in this paper: RA, rheumatoid arthritis; CD62L, L-selectin; PG, proteoglycan (aggrecan); hPG, human cartilage PG; IVM, intravital video microscopy; KO, knockout; LN, lymph node; mPG, mouse PG; PGIA, PG-induced arthritis; PSGL-1, P-selectin glycoprotein ligand-1; WT, wild type.
Received for publication February 21, 2006. Accepted for publication May 18, 2006.
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2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88: 7538-7542. 2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13: 759-769. [Medline]4 integrin in the inflammatory response of neutrophils. J. Exp. Med. 194: 219-226. blockade in patients with rheumatoid arthritis. Arthritis Rheum. 43: 38-47. [Medline]
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3 ankles/score/group) and pooled according to score and genotype. The pooled cells were labeled with anti-Gr-1 mAb and analyzed by flow cytometry. The data shown are from two experiments and indicate the mean percentage of Gr-1+ cells in pooled synovial fluid samples of WT and KO ankle joints.