Functions of Smad Transcription Factors in TGF-ß1-Induced Selectin Ligand Expression on Murine CD4 Th Cells.

Selectins are carbohydrate-binding adhesion molecules that control leukocyte traffic. Induction of selectin ligands on T cells is controlled primarily by cytokines, including TGF-ß1, and requires p38α MAPK, but transcriptional mechanisms that underlie cytokine-driven selectin ligand expression are poorly understood. In this study, we show, using mice with conditional deletions of the TGF-ß1-responsive transcription factors Smad2, Smad3, or Smad4, that induction of selectin ligands on CD4 cells in response to TGF-ß1 requires Smad4 plus either Smad2 or Smad3. Analysis of CD4 cells from mice with only one functional Smad4 allele revealed a sharp gene dosage effect, suggesting the existence of a threshold of TGF-ß1 signal strength required for selectin ligand induction. Both Smad4 plus either Smad2 or Smad3 were selectively required for induction of Fut7 and Gcnt1, glycosyltransferases critical for selectin ligand biosynthesis, but they were not required for St3gal4 or St3gal6 induction. Smad4 plus either Smad2 or Smad3 were also required for induction of Runx transcription factors by TGF-ß1. Enforced expression of Runx2, but not Runx1 or Runx3, in Smad2/Smad3 doubly deficient CD4 cells restored selectin ligand expression to wild-type levels. In contrast, enforced expression of Runx1, Runx2, or Runx3 failed to restore differentiation of TGF-ß1-dependent Th cell lineages, including Th17, Th9, and induced regulatory T cells. These results show that Smads are directly required for Th cell differentiation independent of Runx induction but only indirectly required via Runx2 for TGF-ß1-induced selectin ligand induction on murine CD4 T cells.

Therapeutic inflammatory monocyte modulation using immune-modifying microparticles.

Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3-mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate-induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

Defining the functional boundaries of the murine α1,3-fucosyltransferase Fut7 reveals a remarkably compact locus.

Fut7 encodes an α1,3-fucosyltransferase critical for biosynthesis of glycan ligands for all three selectins. Consistent with this function, Fut7 expression is limited to hematopoietic cells and high endothelial cells which express selectin ligands. Mechanisms that govern Fut7 expression are poorly defined. To begin to understand the molecular genetic basis for transcriptional regulation of Fut7, a transgenic, gain-of-function, genetic complementation approach in mice was used to define the "functional boundaries" of the murine Fut7 locus, defined here as any uninterupted stretch of genomic DNA that contains all cis-acting genetic elements essential for accurate physiologic expression. A 12.7-kb contiguous genomic interval, which lies completely between the highly conserved flanking Npdc1 and Abca2 loci on chromosome 2 and which contains the complete transcriptional unit plus ∼7.4 kb upstream of the transcriptional start site and ∼2 kb downstream of the transcriptional termination and polyadenylation sites, was used as a transgene (Tg) on a Fut7 null background. Tg+ mice exhibited restoration of Fut7 gene expression and physiologic levels of selectin ligand expression and function on neutrophils, activated T cells, and high endothelial cells and corrected the functional defects in these cells found in Fut7 null mice without leading to detectable expression of Fut7 in normally non-expressing tissues. These results demonstrate that all genetic information essential for appropriate and selective expression of Fut7 in diverse cell types and in response to distinct developmental signals is contained within this comparatively small genetic region.

Overlapping roles for endothelial selectins in murine hematopoietic stem/progenitor cell homing to bone marrow.

Selectins are carbohydrate-binding adhesion molecules that are critically involved in leukocyte recognition of endothelium. The endothelial selectins have been implicated in homing of hematopoietic stem and progenitor cells (HSPCs) to the bone marrow (BM) during bone marrow transplant (BMT), but the precise roles of individual selectins in this process have never been defined. BMT of lethally irradiated mice lacking both endothelial selectins (E/P KO) with limiting numbers of wild type BM cells rescued significantly fewer E/P KO than WT recipients, but higher numbers of transplanted WT cells rescued E/P KOs in a dose-dependent fashion. Short-term homing assays confirmed a substantial defect in HSPC homing to BM in E/P KO mice. In contrast, BMT of E-selectin null or P-selectin null mice at limiting cell number uniformly rescued greater than 95% of the transplanted animals. Consistent with these functional results, flow cytometric analysis revealed both E-selectin ligands and P-selectin ligands on distinct subsets of HSPC. These results demonstrate overlapping functions for the endothelial selectins in HSPC homing to BM in the setting of BMT, and define a novel aspect of HSPC heterogeneity linked to selectin ligand expression.

A transgenic mouse model of plasma cell malignancy shows phenotypic, cytogenetic, and gene expression heterogeneity similar to human multiple myeloma.

Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. We have previously shown that the targeted expression of the transgenes c-Myc and Bcl-X(L) in murine plasma cells produces malignancy that displays features of human myeloma, such as localization of tumor cells to the bone marrow and lytic bone lesions. We have isolated and characterized in vitro cultures and adoptive transfers of tumors from Bcl-xl/Myc transgenic mice. Tumors have a plasmablastic morphology and variable expression of CD138, CD45, CD38, and CD19. Spectral karyotyping analysis of metaphase chromosomes from primary tumor cell cultures shows that the Bcl-xl/Myc tumors contain a variety of chromosomal abnormalities, including trisomies, translocations, and deletions. The most frequently aberrant chromosomes are 12 and 16. Three sites for recurring translocations were also identified on chromosomes 4D, 12F, and 16C. Gene expression profiling was used to identify differences in gene expression between tumor cells and normal plasma cells (NPC) and to cluster the tumors into two groups (tumor groups C and D), with distinct gene expression profiles. Four hundred and ninety-five genes were significantly different between both tumor groups and NPCs, whereas 124 genes were uniquely different from NPCs in tumor group C and 204 genes were uniquely different from NPCs in tumor group D. Similar to human myeloma, the cyclin D genes are differentially dysregulated in the mouse tumor groups. These data suggest the Bcl-xl/Myc tumors are similar to a subset of plasmablastic human myelomas and provide insight into the specific genes and pathways underlying the human disease.

Stat3 and Stat4 direct development of IL-17-secreting Th cells.

IL-17-secreting CD4(+) T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFbeta1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFbeta1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORgammat expression in TGFbeta1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFbeta1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types.

Critical role for Stat3 in T-dependent terminal differentiation of IgG B cells.

Stat proteins are latent cytoplasmic transcription factors that are crucial in many aspects of mammalian development. In the immune system, Stat3 has distinct roles in T-cell, neutrophil, and macrophage function, but a role for Stat3 in B-cell development, particularly in the terminal differentiation of B cells into antibody-secreting plasma cells, has never been directly tested. In this study, we used the Cre/lox system to generate a mouse strain in which Stat3 was conditionally deleted in the B-cell lineage (Stat3(fl/fl)CD19(Cre/+)). B-cell development, establishment of the peripheral B-cell compartment, and baseline serum antibody levels were unperturbed in Stat3(fl/fl)CD19(Cre/+) mice. Strikingly, Stat3(fl/fl)CD19(Cre/+) mice displayed profound defects in T-dependent (TD) IgG responses, but normal TD IgM, IgE, and IgA responses and T-independent (TI) IgM and IgG3 responses. In addition, germinal center (GC) formation, isotype switching, and generation of memory B cells, including IgG+ memory cells, were all intact in Stat3(fl/fl)CD19(Cre/+) mice, indicating that the requirement for Stat3 was limited to plasma cell differentiation. These results demonstrate a profound yet highly selective role for Stat3 in TD IgG plasma cell differentiation, and therefore represent a unique example of a transcription factor regulating isotype-specific terminal B-cell differentiation.

H-Ras and phosphoinositide 3-kinase cooperate to induce alpha(1,3)-fucosyltransferase VII expression in Jurkat T cells.

The alpha(1,3)-fucosyltransferase FucT-VII is essential for the biosynthesis of selectin ligands, but the signaling pathways mediating FucT-VII induction in T cells and other lymphocytes are poorly understood. We have shown previously that sustained activation of Ras in Jurkat T cells induces FucT-VII transcription, which requires the Raf-MEK-ERK pathway. In this study we report that FucT-VII induction is specific to the H-Ras isoform. Jurkat T cells retrovirally transduced with constitutively active H-Ras but not N- or K-Ras up-regulated expression of FucT-VII. Pharmacological inhibition studies also revealed that phosphoinositide 3-kinase (PI3K) activity is required for H-Ras-mediated FucT-VII induction. However, the ability of H-Ras to selectively induce FucT-VII is not a function of the inability of the N- or K-Ras isoforms to activate Raf or PI3K pathways. The use of effector-loop domain mutants of H-Ras, which are impaired for their ability to interact selectively with individual effectors alone or in combination with active Raf, indicated that induction of FucT-VII requires the concomitant activation of at least three signaling pathways. These studies show that H-Ras mediates FucT-VII induction in Jurkat T cells via the activation of the Raf, PI3K, and a distinct, H-Ras-specific effector signaling pathway.

Complexity within the plasma cell compartment of mice deficient in both E- and P-selectin: implications for plasma cell differentiation.

Antibody-secreting plasma cells represent the critical end-stage effector cells of the humoral immune response. Here, we show that several distinct plasma cell subsets are concurrently present in the lymph nodes, spleen, and bone marrow of mice deficient in both E- and P-selectin. One of these subsets was a B220-negative immunoglobulin g (IgG) plasma cell population expressing low to negative surface levels of syndecan-1. Examination of the chemotactic responsiveness of IgG plasma cell subsets revealed that migration toward stromal cell-derived factor 1/CXC ligand 12 (SDF-1/CXCL12) was primarily limited to the B220-lo subset regardless of tissue source. Although B220-negative plasma cells did not migrate efficiently in response to CXCL12 or to other chemokines for which receptor mRNA was expressed, these cells expressed substantial surface CXC chemokine receptor-4 (CXCR4), and CXCL12 stimulation rapidly induced extracellular signal regulated kinase 1 (ERK1)/ERK2 phosphorylation, demonstrating that CXCR4 retained signaling capacity. Therefore, B220-negative plasma cells exhibit a selective uncoupling of chemokine receptor expression and signaling from migration. Taken together, our findings document the presence of significant heterogeneity within the plasma cell compartment, which suggests a complex step-wise scheme of plasma cell differentiation in which the degree of differentiation and tissue location can influence the chemotactic responsiveness of IgG plasma cells.

Induction of FucT-VII by the Ras/MAP kinase cascade in Jurkat T cells.

Induction of the alpha1,3-fucosyltransferase FucT-VII in T lymphocytes is crucial for selectin ligand formation, but the signaling and transcriptional pathways that govern FucT-VII expression are unknown. Here, using a novel, highly phorbol myristate acetate (PMA)-responsive variant of the Jurkat T-cell line, we identify Ras and downstream mitogen-activated protein (MAP) kinase pathways as essential mediators of FucT-VII gene expression. PMA induced FucT-VII in only a subset of treated cells, similar to expression of FucT-VII in normal activated CD4 T cells. Introduction of constitutively active Ras or Raf by recombinant retroviruses induced FucT-VII expression only in that subset of cells expressing the highest levels of Ras, suggesting that induction of FucT-VII required a critical threshhold of Ras signaling. Both PMA treatment and introduction of active Ras led to rolling on E-selectin. Pharmacologic inhibition studies confirmed the involvement of the classic Ras-Raf-MEK-extracellular signal-regulated kinase (Ras-Raf-MEK-ERK) pathway in FucT-VII induction by PMA, Ras, and Raf. These studies also revealed a second, Ras-induced, Raf-1-independent pathway that participated in induction of FucT-VII. Strong activation of Ras represents a major pathway for induction of FucT-VII gene expression in T cells.

Sialyltransferase specificity in selectin ligand formation.

Selectin ligands are glycan structures that participate in leukocyte trafficking and inflammation. At least 6 ST3Gal sialyltransferases (I-VI) have been identified that may contribute to selectin ligand formation. However, it is not known which of these sialyltransferases are involved in vivo and whether they may differentially regulate selectin function. We have produced and characterized mice genetically deficient in ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in selectin ligand formation, there was no finding of leukocytosis with these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was found to play a role in the synthesis of selectin ligands. In vitro rolling of marrow-derived neutrophils on E- or P-selectins presented by Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV. However, in a tumor necrosis factor alpha (TNF-alpha)-induced inflammation model in vivo, no defect among P-selectin ligands was observed. Nevertheless, the number of leukocytes rolling on postcapillary venules in an E-selectin-dependent manner was decreased while E-selectin-dependent rolling velocity was increased. We propose that multiple ST3Gal sialyltransferases contribute to selectin ligand formation, as none of these ST3Gal deficiencies recapitulated the degree of E- and P-selectin ligand deficit observed on neuraminidase treatment of intact neutrophils. Our findings indicate a high degree of functional specificity among sialyltransferases and a substantial role for ST3Gal-IV in selectin ligand formation.

The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity.

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.

L-selectin dimerization enhances tether formation to properly spaced ligand.

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.

Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3'-phosphate,5'-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcbeta1-6[Galbeta1-3]GalNAc-pNP (core 2), GlcNAcbeta1-6ManOMe, and GlcNAcbeta1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcbeta1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919-929).