Expression of CD44 and L-selectin in the innate immune system is required for severe joint inflammation in the proteoglycan-induced murine model of rheumatoid arthritis

Proteoglycan (PG)-induced arthritis, a murine model of rheumatoid arthritis, is characterized by autoimmunity against mouse cartilage PG and chronic joint inflammation. L-selectin (CD62L) and CD44 are major adhesion molecules on leukocytes that regulate their homing to lymph nodes and entry into inflamed tissues. In the present study, we studied the requirement for CD44 and CD62L expression for mediating lymphocyte homing, thus permitting the development of autoimmunity vs mediating the entry of leukocytes into the joints, thus allowing inflammation in PG-induced arthritis. We immunized wild-type, CD44 knockout (KO), CD62L KO, and double (CD44/CD62L) KO BALB/c mice with PG and monitored the effects of gene deficiencies on PG-specific immunity, arthritis severity, leukocyte trafficking, and the ability of lymphocytes to adoptively transfer disease to syngeneic SCID mice. Single and double KO mice demonstrated reduced PG-specific spleen cell proliferation, but the production of Th cytokines and autoantibodies was comparable in KO and wild-type mice. KO leukocytes had reduced ability to adhere tightly to the synovial endothelium in arthritic joints. This diminished leukocyte adhesion correlated with the magnitude of granulocyte (neutrophil) influx and the severity of inflammation, which were both reduced in the joints of KO mice. However, transfer of spleen cells from mildly arthritic KO donors to SCID hosts resulted in development of severe arthritis. Our results indicate that CD44 and CD62L expression in the cells of the innate immune system (granulocytes) is important for their efficient influx into the joints and also suggest that granulocytes play a crucial role in arthritis progression.     

Overlapping roles for L-selectin and P-selectin in antigen-induced immune responses in the microvasculature

Although L-selectin mediates lymphocyte attachment to endothelial venules of peripheral lymph nodes, its role in leukocyte recruitment into tissues following Ag challenge is less well established. The objective of this study was to systematically examine the role of L-selectin in leukocyte rolling in the peripheral microvasculature during the first 24 h of an immune response. A type I hypersensitivity response was elicited in wild-type (C57BL/6) and L-selectin-deficient mice by systemic (i.p.) sensitization and intrascrotal challenge with chicken egg OVA. The cremaster microcirculation was observed in untreated and sensitized mice 4, 8, and 24 h post-Ag challenge by intravital microscopy. Leukocyte recruitment in L-selectin-deficient mice and wild-type mice treated with an L-selectin function-blocking mAb was examined at each time point. Ag challenge induced a significant increase in leukocyte rolling (60 cells/min/venule to approximately 300 cells/min/venule) in wild-type mice at 4-24 h. This response was reduced by approximately 60-70% in L-selectin-deficient mice and in wild-type mice treated with an L-selectin-blocking mAb. P-selectin blockade by Ab completely inhibited leukocyte rolling at 4-24 h in wild-type animals and also blocked the residual rolling seen in L-selectin-deficient mice. Blocking E-selectin function had no effect on leukocyte rolling flux at any time point in wild-type or L-selectin-deficient mice. Despite reduced rolling, leukocyte adhesion and emigration were not measurably reduced in the L-selectin-deficient mice in this vascular bed. In conclusion, leukocyte rolling is L-selectin-dependent post-Ag challenge with L-selectin and P-selectin sharing overlapping functions.     

L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo

L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.     

A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

Role of CD44 and hyaluronan in neutrophil recruitment

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44(-/-) and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44(-/-) mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44(-/-) mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44(-/-) mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44(-/-) mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.     

Selectin-independent leukocyte rolling and adhesion in mice deficient in E-, P-, and L-selectin and ICAM-1

To study selectin-independent leukocyte recruitment and the role of intercellular adhesion molecule-1 (ICAM-1), we generated mice lacking all three selectins and ICAM-1 (E/P/L/I-/-) by bone marrow transplantation. These mice were viable and appeared healthy under vivarium conditions, although they showed a 97% reduction in leukocyte rolling, a 63% reduction in leukocyte firm adhesion, and a 99% reduction of neutrophil recruitment in a thioglycollate-induced model of peritonitis at 4 and 24 h. Mononuclear cell recruitment was almost unaffected. All residual leukocyte rolling and most leukocyte adhesion in these mice depended on alpha(4)-integrins, but a small number of leukocytes (6% of wild-type control) still became adherent in the absence of all known rolling mechanisms (E-, P-, L-selectin and alpha(4)-integrins). A striking similarity of leukocyte adhesion efficiency in E/P/L-/- and E/P/I-/- mice suggests a pathway in which leukocyte rolling through L-selectin requires ICAM-1 for adhesion and recruitment. Comparison of our data with mice lacking individual or other combinations of adhesion molecules reveal that elimination of more adhesion molecules further reduces leukocyte recruitment but the effect is less than additive.     

Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow

We demonstrate an additional step and a positive feedback loop in leukocyte accumulation on inflamed endothelium. Leukocytes in shear flow bind to adherent leukocytes through L-selectin/ligand interactions and subsequently bind downstream and roll on inflamed endothelium, purified E-selectin, P-selectin, L-selectin, VCAM-1, or peripheral node addressin. Thus adherent leukocytes nucleate formation of strings of rolling cells and synergistically enhance leukocyte accumulation. Neutrophils, monocytes, and activated T cell lines, but not peripheral blood T lymphocytes, tether to each other through L-selectin. L- selectin is not involved in direct binding to either E- or P-selectin and is not a major counterreceptor of endothelial selectins. Leukocyte- leukocyte tethers are more tolerant to high shear than direct tethers to endothelial selectins and, like other L-selectin-mediated interactions, require a shear threshold. Synergism between leukocyte- leukocyte and leukocyte-endothelial interactions introduces novel regulatory mechanisms in recruitment of leukocytes in inflammation.     

Differential requirements for core2 glucosaminyltransferase for endothelial L-selectin ligand function in vivo

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 beta1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-alpha-treated cremaster muscles and in Peyer's patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2(-/-)) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2(-/-) mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer's patch HEV was not significantly different between core2(-/-) and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2(-/-) mice, but significant rolling in controls. In Peyer's patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2(-/-) mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2(-/-) and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer's patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.     

A functional role for circulating mouse L-selectin in regulating leukocyte/endothelial cell interactions in vivo

L-selectin mediates the initial capture and subsequent rolling of leukocytes along inflamed vascular endothelium and mediates lymphocyte migration to peripheral lymphoid tissues. Leukocyte activation induces rapid endoproteolytic cleavage of L-selectin from the cell surface, generating soluble L-selectin (sL-selectin). Because human sL-selectin retains ligand-binding activity in vitro, mouse sL-selectin and its in vivo relevance were characterized. Comparable with humans, sL-selectin was present in adult C57BL/6 mouse sera at approximately 1.7 micro g/ml. Similar levels of sL-selectin were present in sera from multiple mouse strains, despite their pronounced differences in cell surface L-selectin expression levels. Adhesion molecule-deficient mice prone to spontaneous chronic inflammation and mice suffering from leukemia/lymphoma had 2.5- and 20-fold increased serum sL-selectin levels, respectively. By contrast, serum sL-selectin levels were reduced by 70% in Rag-deficient mice lacking mature lymphocytes. The majority of serum sL-selectin had a molecular mass of 65-75 kDa, consistent with its lymphocyte origin. Slow turnover may explain the relatively high levels of sL-selectin in vivo. The t(1/2) of sL-selectin, assessed by transferring sera from wild-type mice into L-selectin-deficient mice and monitoring serum sL-selectin levels by ELISA, was >20 h, and it remained detectable for longer than 1 wk. Short-term in vivo lymphocyte migration assays demonstrated that near physiologic levels ( approximately 0.9 micro g/ml) of sL-selectin decreased lymphocyte migration to peripheral lymph nodes by >30%, with dose-dependent inhibition occurring with increasing sL-selectin concentrations. These results suggest that sL-selectin influences lymphocyte migration in vivo and that the increased sL-selectin levels present in certain pathologic conditions may adversely affect leukocyte migration.     

Leukocyte arrest during cytokine-dependent inflammation in vivo

Leukocyte rolling along the walls of inflamed venules precedes their adhesion during inflammation. Rolling leukocytes are thought to arrest by engaging beta2 integrins following cellular activation. In vitro studies suggest that chemoattractants may instantaneously activate and arrest rolling leukocytes. However, how leukocytes stop rolling and become adherent in inflamed venules in vivo has remained rather mysterious. In this paper we use a novel method of tracking individual leukocytes through the microcirculation to show that rolling neutrophils become progressively activated while rolling down the venular tree. On average, leukocytes in wild-type mice roll for 86 s (and cover 270 microm) before becoming adherent with an efficiency around 90%. These rolling leukocytes exhibit a gradual beta2 integrin-dependent decrease in rolling velocity that correlates with an increase in intracellular free calcium concentration before arrest. Similar tracking analyses in gene-targeted mice demonstrate that the arrest of rolling leukocytes is very rare when beta2 integrins are absent or blocked by a mAb. Arrest is approximately 50% less efficient in the absence of E-selectin. These data suggest a model of leukocyte recruitment in which beta2 integrins play a critical role in stabilizing leukocyte rolling during a protracted cellular activation period before arrest and firm adhesion.     

Leukocyte rolling velocities and migration are optimized by cooperative L-selectin and intercellular adhesion molecule-1 functions

Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L-selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926-FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L-selectin mAb. In addition, rolling velocities of lymphocytes from CD18-hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18-hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling.     

Direct in vivo monitoring of acute allergic reactions in human conjunctiva

Immediate allergic reactions are initiated by allergen-induced, specific IgE-mediated mast cell degranulation and involve leukocyte recruitment into the inflamed site. We compared conjunctival signs, symptoms, and in vivo leukocyte rolling and extravasation into sites of inflammation in five patients allergic to birch pollen and in 10 nonallergic controls who received a challenge to birch allergen or histamine. Both the specific allergen in allergic patients and histamine, both in patients and in healthy controls, induced symptoms and signs of an immediate allergic reaction together with leukocyte rolling within the conjunctival blood vessels. However, only allergen, not histamine, caused leukocyte extravasation into the site of inflammation in the allergic patients. Allergen also increased expression of endothelial P-selectin in conjunctival vessels and slowed the rolling of leukocytes which is required for their extravasation from blood circulation into the target tissue. Finally, i.v. heparin strongly reduced the number of slowly rolling cells during allergen- or histamine-induced reactions and this can probably hinder the leukocyte extravasation after allergen exposure. These findings suggest that slow rolling is required for leukocyte extravasation in acute allergic reactions, and it can be inhibited by heparin in vivo in therapeutically relevant conditions.     

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L- selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L- selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo- glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.     

Severe impairment of leukocyte recruitment in ppGalNAcT-1-deficient mice

P-selectin glycoprotein ligand-1 plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT-1) initiates core-type protein O-glycosylation. To address whether the glycosylation of P-selectin glycoprotein ligand-1 by ppGalNAcT-1 is important for leukocyte recruitment in vivo, we investigated leukocyte recruitment in untreated and TNF-α-treated cremaster muscles comparing ppGalNAcT-1-deficient mice (Galnt1(-/-)) and wild-type mice. In untreated and TNF-α-treated Galnt1(-/-) mice, leukocyte rolling, adhesion, and transmigration were significantly reduced, with markedly increased rolling velocity compared with control mice. L-selectin-dependent leukocyte rolling was completely abolished in Galnt1(-/-) mice compared with wild-type mice. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT-1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT-1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role for ppGalNAcT-1 in attaching functionally relevant O-linked glycans to selectin ligands.     

CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency

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.     

Leukocyte recruitment and neuroglial activation during facial nerve regeneration in ICAM-1-deficient mice: effects of breeding strategy

Intercellular adhesion molecule 1 (ICAM-1) is a widely expressed glycoprotein involved in leukocyte extravasation and the interaction of lymphocytes with antigen-presenting cells. We examined these aspects of ICAM-1 function in the central nervous system after axonal injury in wild-type and ICAM-1-deficient mice. ICAM-1 immunoreactivity in the normal mouse facial nucleus was restricted to the vascular endothelium. Transection of the facial nerve led to a fast upregulation of ICAM-1 on activated microglia in the axotomized facial nucleus and the infiltration of ICAM-1-positive lymphocytes. Labeling elsewhere was unchanged. In homozygous ICAM-1 mutant mice, ICAM-1 was absent from endothelial cells and lymphocytes, but low levels of ICAM-1 were detected on cell membranes of reactive microglial cells. Comparison of wild-type animals with homozygously bred, ICAM-1-deficient mice showed a reduction of astrocytic and microglial activation, massive late axonal sprouting, and decreased lymphocyte infiltration. These experiments were repeated in F1 progeny of heterozygous mice on a C57BL/6 background. Neuroglial activation and lymphocyte infiltration in F1 homozygously deficient mice was unaffected compared with wild-type siblings. The invading ICAM-1-deficient lymphocytes also adhered to the ICAM-1-positive phagocytotic microglial cells in the ICAM-1 mutants. No change in the recruitment of macrophages and granulocytes into the crushed facial nerve, and no effect on axonal regeneration occurred. These data argue against the requirement of endothelial ICAM-1 in the recruitment of leukocytes into the crushed peripheral nerve or the axotomized facial motor nucleus and stress the importance of adequately matched controls in studying the effects of gene deletion in experimental animals.     

Decreased IgG production but increased MIP-1beta expression in collagen-induced arthritis in C-C chemokine receptor 5-deficient mice

Collagen-induced arthritis (CIA) is a widely used model of human rheumatoid arthritis (RA) characterized by chronic inflammation of the synovial joints. The pathogenesis of RA and CIA has not been completely defined, but both involve the recruitment of leukocytes and lymphocytes to the joints and Th1-type cell mediated autoimmune responses. The C-C chemokine receptor 5 (CCR5) is preferentially expressed on Th1 cells and has been strongly implicated in inflammatory process through trafficking of leukocytes and lymphocytes into the sites of inflammation. We investigated the role of the CCR5 in CIA using CCR5 knockout mice (CCR5-/-) in which we analyzed the consequences of CCR5 deficiency for the immune response and inflammation. We found that CCR5-/- mice showed a significant reduction in the incidence of CIA after collagen II (CII)-immunization as compared to wild-type (CCR5+/+) mice. The reduced incidence seen in CCR5-/- mice was associated with these animals having significantly lower IgG levels, especially IgG2a and IgG2b antibodies against CII, as well as an obviously augmented IL-10 production in splenocytes. Overproduction of MIP-1beta in CCR5-deficient mice after CII-immunization may contribute partially to the occurrence of arthritis.     

Anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, MAS, in experimental models of arthritis

Activation of the renin-angiotensin (Ang) system induces inflammation via interaction between Ang II and type 1 receptor on leukocytes. The relevance of the new arm of the renin-Ang system, namely Ang-converting enzyme-2/Ang-(1-7)/Mas receptor, for inflammatory responses is not known and was investigated in this study. For this purpose, two experimental models were used: Ag-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Male C57BL/6 wild-type or Mas(-/-) mice were subjected to AIA and treated with Ang-(1-7), the Mas agonist AVE 0991, or vehicle. AdIA was performed in female rats that were given AVE 0991 or vehicle. In wild-type mice, Mas protein is expressed in arthritic joints. Administration of AVE 0991 or Ang-(1-7) decreased AIA-induced neutrophil accumulation, hypernociception, and production of TNF-α, IL-1β, and CXCL1. Histopathological analysis showed significant reduction of inflammation. Mechanistically, AVE 0991 reduced leukocyte rolling and adhesion, even when given after Ag challenge. Mas(-/-) mice subjected to AIA developed slightly more pronounced inflammation, as observed by greater neutrophil accumulation and cytokine release. Administration of AVE 0991 was without effect in Mas(-/-) mice subjected to AIA. In rats, administration of AVE 0991 decreased edema, neutrophil accumulation, histopathological score, and production of IL-1β and CXCL1 induced by AdIA. Therefore, activation of Mas receptors decreases neutrophil influx and cytokine production and causes significant amelioration of arthritis in experimental models of arthritis in rats and mice. This approach might represent a novel therapeutic opportunity for arthritis.     

Joint NOD2/RIPK2 signaling regulates IL-17 axis and contributes to the development of experimental arthritis

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-), NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1β, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis.     

CD44 mediated Hyaluronan adhesion of Toxoplasma gondii-infected leukocytes

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix.