P-Selectin glycoprotein ligand 1 (PSGL-1) is a physiological ligand for E-selectin in mediating T helper 1 lymphocyte migration

P-selectin glycoprotein ligand 1 (PSGL-1) is a sialomucin expressed on leukocytes that mediates neutrophil rolling on the vascular endothelium. Here, the role of PSGL-1 in mediating lymphocyte migration was studied using mice lacking PSGL-1. In a contact hypersensitivity model, the infiltration of CD4(+) T lymphocytes into the inflamed skin was reduced in PSGL-1-deficient mice. In vitro-generated T helper (Th)1 cells from PSGL-1-deficient mice did not bind to P-selectin and migrated less efficiently into the inflamed skin than wild-type Th1 cells. To assess the role of PSGL-1 in P- or E-selectin-mediated migration of Th1 cells, the cells were injected into E- or P-selectin-deficient mice. PSGL-1-deficient Th1 cells did not migrate into the inflamed skin of E-selectin-deficient mice, indicating that PSGL-1 on Th1 cells is the sole ligand for P-selectin in vivo. In contrast, PSGL-1-deficient Th1 cells migrated into the inflamed skin of P-selectin-deficient mice, although less efficiently than wild-type Th1 cells. This E-selectin-mediated migration of PSGL-1-deficient or wild-type Th1 cells was not altered by injecting a blocking antibody to L-selectin. These data provide evidence that PSGL-1 on Th1 cells functions as one of the E-selectin ligands in vivo.     

Interactions of human alpha/beta and gamma/delta T lymphocyte subsets in shear flow with E-selectin and P-selectin

We have compared the ability of human alpha/beta and gamma/delta T lymphocytes to adhere to selectin-bearing substrates, an interaction thought to be essential for homing and localization at sites of inflammation. Both T cell populations form rolling adhesions on E- and P-selectin substrates under physiologic flow conditions. Although equivalent to alpha/beta T cells in binding to E-selectin, gamma/delta T cells demonstrated greater ability to adhere to P-selectin that was purified or expressed on the surface of activated, adherent platelets. Under static conditions, 80% of gamma/delta T cells and 53% of alpha/beta T cells formed shear-resistant adhesions to P-selectin, whereas only 30% of gamma/delta and alpha/beta T cells adhered to E- selectin. The enhance ability of gamma/delta T cells to adhere to P- selectin cannot be attributed to differences in expression of the P- selectin glycoprotein ligand (PSGL-1), as all alpha/beta T cells versus approximately 75% of gamma/delta T cells expressed PSGL-1. Both cell populations expressed a similar percentage of the carbohydrate antigens sialyl LewisX and cutaneous lymphocyte-associated antigen. Depletion of lymphocyte populations or T cell clones bearing these oligosaccharides with the monoclonal antibody CSLEX-1 and HECA-452, respectively, resulted in a substantial reduction in adhesion to E-selectin and slight reduction in adhesion to P-selectin under flow conditions. Treatment of cells with an endopeptidase that selectively degrades O- sialomucins such as PSGL-1, abolished P-selectin but not E-selectin adhesion. Removal of terminal sialic acids with neuraminidase or protease treatment of cells abrogated cell adhesion to both selectin substrates. These results provide direct evidence for the presence of distinct E- and P-selectin ligands on T lymphocytes and suggest that gamma/delta T cells may be preferentially recruited to inflammatory sites during the early stages of an immune response when P-selectin is upregulated.     

P-Selectin Glycoprotein Ligand-1 (PSGL-1) on T Helper 1 but Not on T Helper 2 Cells Binds to P-Selectin and Supports Migration into Inflamed Skin

We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin–Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin–binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.     

Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration

P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin-like selectin counterreceptor that binds to P-selectin, E-selectin, and L-selectin. To determine its physiological role in cell adhesion as a mediator of leukocyte rolling and migration during inflammation, we prepared mice genetically deficient in PSGL-1 by targeted disruption of the PSGL-1 gene. The homozygous PSGL-1-deficient mouse was viable and fertile. The blood neutrophil count was modestly elevated. There was no evidence of spontaneous development of skin ulcerations or infections. Leukocyte infiltration in the chemical peritonitis model was significantly delayed. Leukocyte rolling in vivo, studied by intravital microscopy in postcapillary venules of the cremaster muscle, was markedly decreased 30 min after trauma in the PSGL-1-deficient mouse. In contrast, leukocyte rolling 2 h after tumor necrosis factor alpha stimulation was only modestly reduced, but blocking antibodies to E-selectin infused into the PSGL-1-deficient mouse almost completely eliminated leukocyte rolling. These results indicate that PSGL-1 is required for the early inflammatory responses but not for E-selectin-mediated responses. These kinetics are consistent with a model in which PSGL-1 is the predominant neutrophil P-selectin ligand but is not a required counterreceptor for E-selectin under in vivo physiological conditions.     

P-selectin and P-selectin glycoprotein ligand 1 are major determinants for Th1 cell recruitment to nonlymphoid effector sites in the intestinal lamina propria

The recruitment of activated T cell subsets to sites of effector immune responses is mediated by homing receptors induced upon activation in secondary lymphoid tissue. Using an adoptive transfer model, the intestinal recruitment of CD4+ T cells activated with intraperitoneal antigen in complete Freund's adjuvant was examined. The data demonstrate that activated CD4+ T cells recruited to intestinal Peyer's patches (PP) and lamina propria (LP) up-regulate functional P-selectin glycoprotein ligand 1 (PSGL-1). Blockade of IL-12 inhibited functional PSGL-1 expression and reduced PP and LP CD4+ T cell recruitment by >40%. P-selectin blockade reduced LP recruitment of activated cells by 56% without affecting PP recruitment. Studies of mice examined 3 d after adoptive transfer of differentiated T cell subsets revealed that Th1 but not Th2 cells were recruited to small intestine PP and LP. Mucosal addressin cell adhesion molecule blockade reduced Th1 recruitment to PP by 90% and to LP by >72%, whereas P-selectin blockade reduced Th1 recruitment to PP by 18% and Th1 recruitment to LP by 84%. These data suggest that IL-12-induced functional PSGL-1 expression is a major determinant for the recruitment of Th1 effector cells to noninflamed as well as inflamed intestine.     

Eosinophil adhesion to nasal polyp endothelium is P-selectin-dependent

Tissue eosinophilia is a characteristic feature of a number of inflammatory diseases including asthma and nasal polyposis. Eosinophil migration into tissues is controlled in part by interactions between eosinophil adhesion receptors and counter-structures on the vascular endothelium. To determine the receptors used by eosinophils to adhere to vascular endothelium in allergic inflammation we have adapted the Stamper-Woodruff frozen section assay (FSA) to study eosinophil adhesion to nasal polyp endothelium. Immunohistology indicated that intercellular adhesion molecule 1 (ICAM-1), E-selectin and P-selectin were well expressed by nasal polyp endothelium, whereas expression of vascular cell adhesion molecule 1 (VCAM-1) was weak or absent. Unstimulated human peripheral blood eosinophils adhered specifically to nasal polyp endothelium. Adherence was temperature and divalent cation- dependent and saturable at cell densities > 5 x 10(6) cells/ml. Eosinophil adhesion was almost completely inhibited by a monoclonal antibody (mAb) against P-selectin and by a chimeric molecule consisting of the Fc portion of human IgG and the lectin binding domain of P- selectin, which binds to the P-selectin ligand on leucocytes. Anti-Mac- 1 mAb partially inhibited eosinophil adhesion whereas mAb against E- selectin, L-selectin, ICAM-1, VCAM-1, very late activation antigen 4, and lymphocyte function-associated antigen 1 had no effect. P-selectin is stored in intracellular granules within the endothelial cell and in vitro is only transiently expressed. To determine if P-selectin was expressed on the membrane of the nasal polyp endothelium we compared P- selectin expression in normal skin and nasal polyps after acetone fixation, which permeabilizes cells, and paraformaldehyde, which only allows staining of membrane expressed receptors. In the skin, good expression was seen with acetone fixation but no expression was seen after paraformaldehyde treatment, whereas in nasal polyps, similar expression was observed with both fixatives. In addition immunofluorescence with confocal microscopy demonstrated lumenal staining of nasal polyp endothelium indicating that P-selectin was located on the surface of endothelial cells while in skin only an intracellular granular distribution was apparent. Lastly, whereas eosinophils bound consistently to nasal polyp endothelium, no binding was observed to blood vessels in normal skin further supporting the idea that eosinophils were binding to membrane expressed and not intracellular P-selectin. The importance of P-selectin in eosinophil adhesion to nasal polyp endothelium suggests that P-selectin antagonists may be effective at inhibiting eosinophil accumulation at sites of allergic inflammation.     

Fuc-TVII is required for T helper 1 and T cytotoxic 1 lymphocyte selectin ligand expression and recruitment in inflammation, and together with Fuc-TIV regulates naive T cell trafficking to lymph nodes

To determine how the alpha(1,3)fucosyltransferases Fuc-TIV and Fuc-TVII, and the selectin ligands they control may contribute to the adaptive immune response, contact hypersensitivity (CHS) was characterized in mice deficient in either or both enzymes. We find a substantial CHS deficiency in Fuc-TVII(-/-) mice, and a complete deficiency in Fuc-TIV(-/-)/Fuc-TVII(-/-) mice. These defects are not accounted for by alterations in the number or function of epidermal Langerhans cells required for cutaneous antigen processing and presentation. By contrast, defective CHS in Fuc-TVII(-/-) mice or Fuc-TIV(-/-)/Fuc-TVII(-/-) mice is attributed in part to prominent, or nearly complete deficiencies, respectively, in the complement of naive T lymphocytes available in lymph nodes for antigen-dependent activation, expansion, differentiation, and dissemination. Fuc-TVII deficiency also deletes expression of E- and P-selectin ligands by Th1 and T cytotoxic 1 (Tc1) lymphocytes, annuls T cell trafficking to inflamed cutaneous sites in vivo, and thereby controls an essential component of the efferent phase of the cutaneous immune response. These observations indicate that collaborative contributions of Fuc-TIV and Fuc-TVII to L-selectin ligand synthesis, and to lymphocyte recruitment, are requisite components of the primary cellular immune response, and assign an essential role to Fuc-TVII in control of E- and P-selectin ligand expression by Th1 and Tc1 lymphocytes.     

Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues.

Effector and memory T cells can be subdivided based on their ability to traffic through peripheral tissues such as inflamed skin and intestinal lamina propria, a property controlled by expression of 'tissue-specific' adhesion and chemoattractant receptors. However, little is known about the development of these selectively homing T cell subsets, and it is unclear whether activation in cutaneous versus intestinal lymphoid organs directly results in effector/memory T cells that differentially express adhesion and chemoattractant receptors targeting them to the corresponding nonlymphoid site. We define two murine CD4(+) effector/memory T cell subsets that preferentially localize in cutaneous or intestinal lymphoid organs by their reciprocal expression of the adhesion molecules P-selectin ligand (P-lig) and alpha 4 beta 7, respectively. We show that within 2 d of systemic immunization CD4(+) T cells activated in cutaneous lymph nodes upregulate P-lig, and downregulate alpha 4 beta 7, while those responding to antigen in intestinal lymph nodes selectively express high levels of alpha 4 beta 7 and acquire responsiveness to the intestinal chemokine thymus-expressed chemokine (TECK). Thus, during an immune response, local microenvironments within cutaneous and intestinal secondary lymphoid organs differentially direct T cell expression of these adhesion and chemoattractant receptors, targeting the resulting effector T cells to the inflamed skin or intestinal lamina propria.     

Bovine gamma/delta T cells bind E-selectin via a novel glycoprotein receptor: first characterization of a lymphocyte/E-selectin interaction in an animal model

E-Selectin is an inducible adhesion protein expressed by endothelial cells and recognized by leukocytes during their extravasation from the blood into inflamed tissues. Originally, E-selectin was defined as a myeloid cell-specific adhesion protein, but recent studies have shown it to be recognized by human lymphocytes as well. These lymphocytes represent a memory T cell subset and have been shown to express the HECA-452 carbohydrate epitope (CLA+ lymphocytes). We extend these findings and show that ruminant gamma/delta T cells bind E-selectin as well; and we provide preliminary evidence that this interaction is mediated by a novel glycoprotein receptor on the lymphocyte. Unlike conventional T cells (alpha/beta T cells), gamma/delta T cells from neonatal and mature animals bind E-selectin, suggesting that prior antigen stimulation and differentiation to a memory lymphocyte are not required for this interaction. Neuraminidase treatment of the gamma/delta T cells or addition of ethylenediaminetetraacetic acid (EDTA) to the assay abrogates binding, demonstrating the importance of sialic acid and divalent cations, which is consistent with other E- selectin-mediated adhesion events. However, previously defined E- selectin carbohydrate ligands, such as sialyl Lewis x on neutrophils and the HECA-452 epitope on human memory lymphocytes, are antigenically different than the carbohydrates on ruminant gamma/delta T cells since the mAbs CSLEX and HECA-452 do not recognize these cells. Protease treatment of gamma/delta T cells significantly inhibits their binding to E-selectin; however, previously characterized adhesion glycoproteins, such as L-selectin, CD44, and CD18, are not involved in the adhesive event. An E-selectin affinity column purifies a single glycoprotein of 250 kD (280 kD under reducing conditions) from gamma/delta T cell detergent lysates. Neuraminidase digestion of the 250-kD product as well as EDTA abolishes binding to E-selectin. Finally, E-selectin expression in vivo appears to mediate gamma/delta T cell accumulation. Stimulation of bovine skin with tumor necrosis factor alpha induced an increase in E-selectin expression that was associated with an influx of gamma/delta T cells at the same site.     

P-selectin glycoprotein ligand-1 mediates L-selectin-dependent leukocyte rolling in venules

Leukocyte rolling in postcapillary venules of inflamed tissues is reduced in L-selectin-deficient mice and mice treated with L-selectin blocking antibodies, but the glycoprotein ligand for L-selectin in inflamed venules is unknown. Here, we show that L-selectin-dependent rolling after P-selectin blockade is completely absent in P-selectin glycoprotein ligand-1 (PSGL-1)-/- mice or wild-type mice treated with a PSGL-1 blocking monoclonal antibody. Immunohistochemistry and flow cytometry failed to show PSGL-1 expression on resting or inflamed endothelium or on platelets. To investigate whether leukocyte-expressed PSGL-1 is mediating L-selectin-dependent rolling, we reconstituted lethally irradiated wild-type mice with PSGL-1-/- bone marrow cells. These chimeric mice showed no L-selectin-dependent rolling, suggesting that leukocyte-expressed PSGL-1 mediates L-selectin-dependent rolling. Frame-to-frame video analysis of L-selectin-dependent rolling in wild-type mice showed that the majority of observed L-selectin-dependent leukocyte rolling was between free flowing leukocytes and already adherent leukocytes or possibly leukocyte fragments, followed by E-selectin-dependent leukocyte rolling along the endothelium. Leukocyte rolling was significantly slower for leukocyte-endothelial than leukocyte-leukocyte interactions. We conclude that leukocyte-expressed PSGL-1 serves as the main L-selectin ligand in inflamed postcapillary venules. L-selectin binding to PSGL-1 initiates tethering events that enable L-selectin-independent leukocyte-endothelial interactions. These findings provide a molecular mechanism for the inflammatory defects seen in L-selectin-deficient mice.     

CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin.

The chemokine thymus and activation-regulated chemokine (TARC; CCL17) is displayed by cutaneous (but not intestinal) venules, and is thought to trigger vascular arrest of circulating skin homing memory T cells, which uniformly express the TARC receptor CC chemokine receptor (CCR)4. Cutaneous T cell-attracting chemokine (CTACK; CCL27), expressed by skin keratinocytes, also attracts cutaneous memory T cells, and is hypothesized to assist in lymphocyte recruitment to skin as well. Here we show that chronic cutaneous inflammation induces CD4 T cells expressing E-selectin binding activity (a marker of skin homing memory cells) in draining lymph node, and that these E-selectin ligand+ T cells migrate efficiently to TARC and to CTACK. In 24 h in vivo homing assays, stimulated lymph node T cells from wild-type mice or, surprisingly, from CCR4-deficient donors migrate efficiently to inflamed skin; and an inhibitory anti-CTACK antibody has no effect on wild-type lymphocyte recruitment. However, inhibition with anti-CTACK monoclonal antibody abrogates skin recruitment of CCR4-deficient T cells. We conclude that CTACK and CCR4 can both support homing of T cells to skin, and that either one or the other is required for lymphocyte recruitment in cutaneous delayed type hypersensitivity.     

Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified sphingosine-1-phosphate (S1P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S1P.The thymus does not contain self-renewing T cell progenitors (TCPs) and, therefore, requires continuous importation of progenitors from the blood to sustain T cell output (1, 2). Recruitment of TCPs to the thymus is facilitated by a multistep adhesion cascade initiated by the interaction of P-selectin glycoprotein ligand 1 (PSGL-1) expressed on TCPs, with P-selectin (CD62P) expressed on thymic endothelium (3, 4). Specific N-terminal carbohydrate modifications on PSGL-1 catalyzed by glycosyltransferases are required for binding to P-selectin. In particular, the activity of the core 2 β1,6-glucosaminyltransferase-I (C2GnT1) has been highlighted in selectin ligand formation on PSGL-1 (5). Interaction of PSGL-1 with endothelial P-selectin slows down TCPs, and allows them to respond to local chemokine gradients and to engage the endothelial adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) via integrins, leading to a firm arrest of TCPs on the thymic endothelium (4).Thymic TCP importation is independent of the numbers of available TCPs in the blood (6) and is not a steady-state process, as the thymus alternates between nonreceptive and receptive periods (2). This suggests that a feedback mechanism regulates expression of recruitment signals on vascular or perivascular cells. One factor proposed to control progenitor entry into the thymus is the limited availability of intrathymic niches for which TCPs compete and, thus, gain access to survival and differentiation factors necessary for their development (6). Full thymic niches are thought to trigger a feedback signal to reduce TCP importation, but the nature of these feedback signals and how they influence thymic TCP entry is unknown.To explore the basis of feedback signals that determine periodicity in thymic TCP receptivity, we analyzed the expression of adhesion molecules in WT mice and mouse strains with distinct thymic receptivities. We found that thymic P-selectin and CCL25 expression correlated with thymic TCP receptivity, suggesting that these molecules function as thymic gatekeepers. Furthermore, we found that P-selectin expression and thymic TCP receptivity correlated inversely with peripheral lymphocyte numbers and could be manipulated by experimentally altering peripheral lymphocyte numbers. These results show that the peripheral lymphocyte pool feeds back to thymic TCP entry sites and modifies thymic TCP importation by regulating P-selectin and CCL25 expression.     

Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells.

Different T cell subsets exhibit distinct capacities to migrate into peripheral sites of inflammation, and this may in part reflect differential expression of homing receptors and chemokine receptors. Using an adoptive transfer approach, we examined the ability of functionally distinct subsets of T cells to home to a peripheral inflammatory site. The data directly demonstrate the inability of naive T cells and the ability of effector cells to home to inflamed peritoneum. Furthermore, interleukin (IL)-12 directs the differentiation of either CD4(+) or CD8(+) T cells into effector populations that expresses functional E- and P-selectin ligand and that are preferentially recruited into the inflamed peritoneum compared with T cells differentiated in the presence of IL-4. Recruitment can be blocked by anti-E- and -P-selectin antibodies. The presence of antigen in the peritoneum promotes local proliferation of recruited T cells, and significantly amplifies the Th1 polarization of the lymphocytic infiltrate. Preferential recruitment of Th1 cells into the peritoneum is also seen when cytokine response gene 2 (CRG-2)/interferon gamma-inducible protein 10 (IP-10) is used as the sole inflammatory stimulus. We have also found that P-selectin binds only to antigen-specific T cells in draining lymph nodes after immunization, implying that both antigen- and cytokine-mediated signals are required for expression of functional selectin-ligand.     

P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin under flow. The glycoproteins that enable leukocyte tethering to or rolling on E-selectin are not known. We used gene targeting to prepare PSGL-1-deficient (PSGL-1-/-) mice, which were healthy but had moderately elevated total blood leukocytes. Fluid-phase E-selectin bound to approximately 70% fewer sites on PSGL-1-/- than PSGL-1+/+ neutrophils. Compared with PSGL-1+/+ leukocytes, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in vitro, because their initial tethering to E-selectin was impaired. The residual cells that tethered rolled with the same shear resistance and velocities as PSGL-1+/+ leukocytes. Compared with PSGL-1+/+ mice, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in TNF-alpha-treated venules of cremaster muscle in which P-selectin function was blocked by an mAb. The residual PSGL-1-/- leukocytes that tethered rolled with slow velocities equivalent to those of PSGL-1+/+ leukocytes. These results reveal a novel function for PSGL-1 in tethering leukocytes to E-selectin under flow.     

Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.     

Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions.

Both P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin are localized on the microvilli of neutrophils and have been implicated in the attachment of neutrophils to P-selectin or E-selectin. We directly compared the attachment and rolling of neutrophils on P-selectin and E-selectin under flow, with emphasis on the functions of PSGL-1 and L-selectin. Flowing neutrophils attached more avidly and rolled at lower velocities on P-selectin than on E-selectin at matched densities. Studies with purified molecules indicated that P-selectin and E-selectin bound to a related site on PSGL-1 that overlapped the epitope for the anti-PSGL-1 mAb PL1. E-selectin bound with lower affinity than P-selectin to this site and also bound to an additional site(s) on PSGL-1.PL1 abolished adhesion of neutrophils to P-selectin under shear or static conditions, whereas DREG-56, a mAb to L-selectin, had no effect on adhesion to P-selectin. PL1 inhibited attachment of neutrophils to E-selectin under flow but not static conditions. DREG-56 also inhibited attachment of flowing neutrophils to E-selectin, and a combination of DREG-56 and PL1 nearly eliminated attachment to E-selectin under flow. These data suggest that PSGL-1 functions cooperatively with L-selectin to mediate optimal attachment of flowing neutrophils to E-selectin but not to P-selectin. Neutrophils attach more efficiently and with greater strength to P-selectin, perhaps because of the higher affinity of P-selectin for the PL1-defined site on PSGL-1.     

ICOS-dependent extrafollicular helper T cells elicit IgG production via IL-21 in systemic autoimmunity

The role of specialized follicular helper T (T_FH) cells in the germinal center has become well recognized, but it is less clear how effector T cells govern the extrafollicular response, the dominant pathway of high-affinity, isotype-switched autoantibody production in the MRL/MpJ-Fas^lpr (MRL^lpr) mouse model of lupus. MRL^lpr mice lacking the Icos gene have impaired extrafollicular differentiation of immunoglobulin (Ig) G⁺ plasma cells accompanied by defects in CXC chemokine receptor (CXCR) 4 expression, interleukin (IL) 21 secretion, and B cell helper function in CD4 T cells. These phenotypes reflect the selective loss of a population of T cells marked by down-regulation of P-selectin glycoprotein ligand 1 (PSGL-1; also known as CD162). PSGL-1^lo T cells from MRL^lpr mice express CXCR4, localize to extrafollicular sites, and uniquely mediate IgG production through IL-21 and CD40L. In other autoimmune strains, PSGL-1^lo T cells are also abundant but may exhibit either a follicular or extrafollicular phenotype. Our findings define an anatomically distinct extrafollicular population of cells that regulates plasma cell differentiation in chronic autoimmunity, indicating that specialized humoral effector T cells akin to T_FH cells can occur outside the follicle.     

Adhesion molecules as therapeutic targets for autoimmune diseases and transplant rejection

Inflammatory disorders such as autoimmune diseases and graft rejection are mediated by activated leukocytes, particularly T lymphocytes, which penetrate the inflamed tissue and perpetuate or amplify the immune reaction. In an unstimulated state, leukocytes do not readily adhere to the vascular endothelium. However, inflammatory signals induce the expression of proteins on the endothelial cell surface that promote the adhesion and extravasation of activated immune cells from the circulation into the underlying tissues. Key among these molecules are P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cells, and their respective counter receptors, P-selectin glycoprotein ligand-1 (PSGL-1), leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4), on the leukocytes. In vitro blockade of these molecules inhibits the adhesion of leukocytes. In many cases there is attenuation of leukocyte activation as well. Adhesion blockade in animal models prevents or ameliorates graft rejection and disease severity in autoimmune models. Clinical studies with humanised monoclonal antibodies which interfere with LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions have shown significant efficacy and good safety profiles in autoimmune disease, including psoriasis, multiple sclerosis and inflammatory bowel disease. Thus, adhesion blockade is emerging as a useful therapeutic strategy in several inflammatory settings.     

Differential regulation of human and murine P-selectin expression and function in vivo

Leukocytes roll on P-selectin after its mobilization from secretory granules to the surfaces of platelets and endothelial cells. Tumor necrosis factor (TNF), IL-1β, and lipopolysaccharide increase synthesis of P-selectin in murine but not in human endothelial cells. To explore the physiological significance of this difference in gene regulation, we made transgenic mice bearing the human Selp gene and crossed them with mice lacking murine P-selectin (Selp(-/-)). The transgenic mice constitutively expressed human P-selectin in platelets, endothelial cells, and macrophages. P-selectin mediated comparable neutrophil migration into the inflamed peritoneum of transgenic and wild-type (WT) mice. Leukocytes rolled similarly on human or murine P-selectin on activated murine platelets and in venules of the cremaster muscle subjected to trauma. However, TNF increased murine P-selectin in venules, slowing rolling and increasing adhesion, whereas it decreased human P-selectin, accelerating rolling and decreasing adhesion. Both P- and E-selectin mediated basal rolling in the skin of WT mice, but E-selectin dominated rolling in transgenic mice. During contact hypersensitivity, murine P-selectin messenger (m) RNA was up-regulated and P-selectin was essential for leukocyte recruitment. However, human P-selectin mRNA was down-regulated and P-selectin contributed much less to leukocyte recruitment. These findings reveal functionally significant differences in basal and inducible expression of human and murine P-selectin in vivo.     

CD44 is a physiological E-selectin ligand on neutrophils

The selectin family of adhesion molecules and their glycoconjugated ligands are essential for blood polymorphonuclear neutrophil (PMN) extravasation into inflammatory and infectious sites. However, E-selectin ligands on PMNs are not well characterized. We show here that CD44 immunopurified from G-CSF-differentiated 32D cells or from peripheral blood PMNs binds specifically to E-selectin. In contrast, CD44 extracted from bone marrow stromal or brain endothelial cell lines does not interact with E-selectin, suggesting cell-specific posttranslational modifications of CD44. PMN-derived CD44 binding activity is mediated by sialylated, alpha(1,3) fucosylated, N-linked glycans. CD44 enables slow leukocyte rolling on E-selectin expressed on inflamed endothelium in vivo and cooperates with P-selectin glycoprotein ligand-1 to recruit neutrophils into thioglycollate-induced peritonitis and staphylococcal enterotoxin A-injected skin pouch. CD44 extracted from human PMNs also binds to E-selectin. Moreover, we demonstrate that CD44 is hypofucosylated in PMNs from a patient with leukocyte adhesion deficiency type II, suggesting that it contributes to the syndrome. These findings thus suggest broader roles for CD44 in the innate immune response and uncover a potential new target for diseases in which selectins play a prominent role.