Hermann J. Ziltener

Cutting Edge: CD43 Functions as a T Cell Counterreceptor for the Macrophage Adhesion Receptor Sialoadhesin (Siglec-1)

Sialoadhesin (Siglec-1) is a macrophage-restricted sialic acid-binding receptor that mediates interactions with hemopoietic cells, including lymphocytes. In this study, we identify sialoadhesin counterreceptors on T lymphocytes. Several major glycoproteins (85, 130, 240 kDa) were precipitated by sialoadhesin-Fc fusion proteins from a murine T cell line (TK-1). Binding of sialoadhesin to these glycoproteins was sialic acid dependent and was abolished by mutation of a critical residue (R97A) of the sialic acid binding site in the membrane distal Ig-like domain of sialoadhesin. The 130- and 240-kDa sialoadhesin-binding glycoproteins were identified as the sialomucins CD43 and P-selectin glycoprotein ligand 1 (CD162), respectively. CD43 expressed in COS cells supported increased binding to immobilized sialoadhesin. Finally, sialoadhesin bound different glycoforms of CD43 expressed in Chinese hamster ovary cells, including unbranched (core 1) and branched (core 2) O-linked glycans, that are normally found on CD43 in resting and activated T cells, respectively. These results identify CD43 as a T cell counterreceptor for sialoadhesin and suggest that in addition to its anti-adhesive role CD43 may promote cell-cell interactions.

PSGL‐1 function in immunity and steady state homeostasis

Summary:  The substantial importance of P‐selectin glycoprotein ligand 1 (PSGL‐1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL‐1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin‐PSGL‐1 interaction requires branched O‐glycan extensions on specific PSGL‐1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O‐glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL‐1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O‐glycans on PSGL‐1 and provide the molecular interactions responsible for the velcro‐like catch bonds that support leukocyte rolling. Such binding of PSGL‐1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL‐1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T‐cell homing to secondary lymphoid organs. PSGL‐1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O‐glycan modifications of PSGL‐1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL‐1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T‐cell traffic. The multi‐level influence of PSGL‐1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O‐glycans. However, central as specific O‐glycosylation is to PSGL‐1 function, in vivo regulation of PSGL‐1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL‐1 glycosylation and to update understanding of PSGL‐1 functional scope.

Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs

P-selectin glycoprotein ligand 1 (PSGL-1) is central to the trafficking of immune effector cells to areas of inflammation through direct interactions with P-selectin, E-selectin and L-selectin. Here we show that PSGL-1 was also required for efficient homing of resting T cells to secondary lymphoid organs but functioned independently of selectin binding. PSGL-1 mediated an enhanced chemotactic T cell response to the secondary lymphoid organ chemokines CCL21 and CCL19 but not to CXCL12 or to inflammatory chemokines. Our data show involvement of PSGL-1 in facilitating the entry of T cells into secondary lymphoid organs, thereby demonstrating the bifunctional nature of this molecule.

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.

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells

The glycosyltransferase core 2 β1–6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8+ T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8+ cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.

Absence of CD43 fails to alter T cell development and responsiveness

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43null mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43null lymph node cells compared with littermate HY-CD43+/− lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43null mice have been based on mice with a mixed 129×C57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43null littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43+ and CD43null littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.

Lymphocytes in the Peritoneum Home to the Omentum and Are Activated by Resident Dendritic Cells

The omentum is of interest in the context of obesity-related metabolic disease where adipose tissue exhibits inflammatory changes; however, the immunology of the omentum is underexplored. The greater omentum is draped from the stomach and consists predominantly of adipose tissue studded with lymphoreticular aggregations (milky spots) that distinguish it from other visceral adipose tissues. Milky spots are thought to contain and conduct leukocytes in transit from the blood to the peritoneal cavity, particularly during peritonitis. We show here that both B and T lymphocytes counterflow from the peritoneal cavity to the omentum in mice. Residence in the omentum was brief with a t1/2 residence time of 6 h. Omentum access was pertussis toxin-sensitive, dependent on activation of the Rap1 GTPase, and on the integrin LFA-1. B cells and CD44high T cells accessed the omentum most efficiently, but homing of resting CD44low T cells was also observed. Omental tissue from normal healthy mice was found to contain CD8−CD11bhighMHC class IIhighCD11chigh dendritic cells that promoted the rapid activation of T cells entering the omentum and cross-presented soluble OVA or OVA acquired from either OVA-expressing Escherichia coli or OVA-pulsed spleen cells. We conclude that the omentum incorporates two key features of immunological sentinel function, actively supported lymphocyte traffic and dendritic cells, that reinforce a conceptual framework for function in stimulating adaptive immunity. These results extend basic understanding of omental and peritoneal cavity immunology and of how proinflammatory events occurring within the peritoneal cavity might affect adipocyte and hepatocyte metabolism.

The Metalloprotease-Disintegrin ADAM8 Is Essential for the Development of Experimental Asthma

RATIONALE Expression of the metalloprotease ADAM8 is increased in patients with asthma, but the functional significance of elevated ADAM8 expression in the context of asthma pathogenesis remains elusive. OBJECTIVES To study development of asthma in ADAM8-deficient mice. METHODS Ovalbumin-induced asthma was studied in wild-type, ADAM8-deficient, and ADAM8-chimeric mice. Lung inflammation was assessed by histology, analysis of bronchoalveolar lavage, and airway hyperresponsiveness. MEASUREMENTS AND MAIN RESULTS ADAM8-deficient mice are highly resistant to the development of ovalbumin-induced airway inflammation and hyperresponsiveness. ADAM8 expression was induced in both hematopoietic cells and the nonhematopoietic microenvironment after induction of asthma, and ADAM8 expression in both cell populations was required for the full manifestation of asthma. Interestingly, loss of ADAM8 on T cells alone was sufficient to significantly decrease the asthma response. The attenuated response was not due to an intrinsic defect in antigen presentation or cytokine production but reflected an impaired migration of T cells, eosinophils, CD11b(+) CD11c(-), and CD11c(+) cells from blood vessels to the lung and alveolar space, suggesting a general hematopoietic cell deficiency in the absence of ADAM8. CONCLUSIONS The results show that ADAM8 plays a proinflammatory role in airway inflammation. The milder disease outcome in the absence of ADAM8 suggests that this protein might be an interesting new target in treatment of this, and potentially other, inflammatory diseases in which recruitment of inflammatory cells is an essential part of pathogenesis.

Mycobacterium tuberculosis employs Cpn60.2 as an adhesin that binds CD43 on the macrophage surface.

CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43‐Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab′)2 antibody‐mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43+/+ versus CD43−/− macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43‐dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone.

CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-α-mediated apoptosis

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43−/− macrophages, priming of cells with IFN‐γ returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43−/− macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF‐α, IL‐12 and IL‐6 by M. tuberculosis‐infected macrophages. We also found that infected resting, but not activated CD43−/− macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro‐inflammatory cytokine TNF‐α restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro‐inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.