Espen S. Baekkevold

Molecular Characterization of NF-HEV, a Nuclear Factor Preferentially Expressed in Human High Endothelial Venules

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyerss patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.

Regional specialization in the mucosal immune system: what happens in the microcompartments?

Mucosal immunity is an important arm of the immune system because it operates in tissues involved in everyday infectious defence as well as in tolerance against innocuous environmental and dietary antigens. Here, Per Brandtzaeg and colleagues discuss compartmentalized regulation of mucosal B cells and mechanisms that might explain the strikingly regionalized effector disparity of the human mucosal immune system.

Naive T Cell Recruitment to Nonlymphoid Tissues: A Role for Endothelium-Expressed CC Chemokine Ligand 21 in Autoimmune Disease and Lymphoid Neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP+ cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP+ cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 ± 10%) and ulcerative colitis (66 ± 1%) expressed CCL21, and many perivascular CD45RA+ naive T cells were found in these tissues, but not in psoriasis, where CCL21+ vessels were rare (17 ± 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.

B cell attracting chemokine 1 (CXCL13) and its receptor CXCR5 are expressed in normal and aberrant gut associated lymphoid tissue

Background and aims: In mice, the B lymphocyte chemoattractant (BLC) CXC chemokine ligand 13 (CXCL13) is sufficient to induce a series of events leading to the formation of organised lymphoid tissue. Its receptor, CXCR5, is required for normal development of secondary lymphoid tissue. However, the human counterpart, B cell attracting chemokine 1 (BCA-1) has only been detected in the stomach and appendix and not in other parts of normal or diseased gut. Hence to elucidate the potential role of this chemokine and its receptor in human gut associated lymphoid tissue (GALT), we analysed their expression in normal intestine and ulcerative colitis (UC). Methods: Frozen sections of surgical specimens were studied by multicolour immunofluorescence staining, in situ mRNA hybridisation, and reverse transcription-polymerase chain reaction. Results: BCA-1 mRNA was detected in all normal colonic and UC specimens. BCA-1 was produced and accumulated in relation to peripheral dendritic elements of lymphoid follicles in Peyers patches and normal colon, as well as in irregular lymphoid aggregates in UC lesions. BCA-1 was partially associated with the traditional follicular dendritic cell phenotype but also with extracellular fibrils in GALT structures. CXCR5 protein was expressed by mantle zone B cells and appeared at a high level on scattered germinal centre T cells. Conclusions: BCA-1 and CXCR5 are expressed in normal GALT structures as well as in irregular lymphoid aggregates in UC. This strongly suggests that BCA-1 plays an important role not only in the formation of normal GALT but also in the generation of aberrant lymphoid tissue in inflammatory bowel disease.

Heterogeneity of Endothelial Cells: The Specialized Phenotype of Human High Endothelial Venules Characterized by Suppression Subtractive Hybridization

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.

From B to A the mucosal way.

Generation of intestinal secretory IgA depends on antigen induction of B cells in organized GALT. A recent paper in Nature reports that in mice the lamina propria provides signals that direct mucosal B cells to undergo Cα class switching and as a basis for SIgA production.

A role for CCR4 in development of mature circulating cutaneous T helper memory cell populations.

Expression of the chemokine receptor CCR4 is strongly associated with trafficking of specialized cutaneous memory T helper (Th) lymphocytes to the skin. However, it is unknown whether CCR4 itself participates in the development of cutaneous Th populations. We have addressed this issue via competitive bone marrow (BM) reconstitution assays; equal numbers of BM cells from CCR4+/+ and CCR4−/− donors were allowed to develop side-by-side within RAG-1−/− hosts. Cells from both donor types developed equally well into B cells, naive CD8 T cells, naive CD4 T cells, interferon-γ+ Th1 cells, and interleukin-4+ Th2 cells. In marked contrast, circulating cutaneous memory Th cells (i.e., E-selectin ligand+ [E-lig+]) were more than fourfold more likely to be derived from CCR4+/+ donors than from CCR4−/− donors. Most of this effect resides within the CD103+ subset of the E-lig+ Th population, in which donor CCR4+/+ cells can outnumber CCR4−/− cells by >12-fold. No similar effect was observed for α4β7+ intestinal memory Th cells or CD103+/E-lig− Th cells. We conclude that CCR4 expression provides a competitive advantage to cutaneous Th cells, either by participating in their development from naive Th cells, or by preferentially maintaining them within the memory population over time.

Sulfation in high endothelial venules: cloning and expression of the human PAPS synthetase

High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. Studies with chlorate, a metabolic inhibitor of sulfation, had previously revealed that production of PAPS (3′‐phosphoadenosine‐5′‐phosphosulfate), the high‐energy donor of sulfate, is required for sulfation and high‐affinity recognition of HEV sialomucins GlyCAM‐1 and CD34 by the lymphocyte homing receptor L‐selectin. Here, we report the molecular characterization of a novel 2.5 kb human cDNA from MECA‐79+ HEV‐derived endothelial cells that encodes the target of chlorate, PAPS synthetase, a multifunctional enzyme containing domains for both ATP sulfurylase and adenosine‐5′‐phosphosulfate kinase. Functional expression of the isolated cDNA in Chinese hamster ovary cells results in high levels of PAPS synthesis, which is abolished by treatment of the transfected cells with chlorate. Northern blot analysis reveals a wide tissue distribution of PAPS synthetase mRNA in the human body, suggesting that human PAPS synthetase may be important for sulfation not only of HEV sialomucins, but also of many other molecules, including mucins such as the P‐selectin ligand PSGL‐1, proteoglycans, hormones, neurotransmitters, drugs, and xenobiotics.

The short form of TSLP is constitutively translated in human keratinocytes and has characteristics of an antimicrobial peptide.

Thymic stromal lymphopoietin (TSLP) has multifaceted immunological functions ranging from maintenance of tolerance to induction of disease. Two human transcript variants of TSLP are described: a long form (variant 1; lfTSLP) consisting of four exons and an alternative, short form (variant 2; sfTSLP) that lacks two exons compared with variant 1. SfTSLP has not been described at the protein level or functionally studied. Here, we demonstrate that the human sfTSLP is the predominant form of TSLP, constitutively expressed at the mRNA and protein level in keratinocytes of oral mucosa and skin and in salivary glands, is released in saliva, and is not regulated in the same manner as the long form. Compared with lfTSLP, sfTSLP exhibits a markedly stronger antibacterial activity. Synthetic sfTSLP did not activate signal transducer and activator of transcription 5 (STAT5) signaling in CD1c+ dendritic cells nor interfered with STAT5 activation by lfTSLP. SfTSLP may, therefore, act as an antimicrobial peptide in the oral cavity and on the skin to create a defense barrier that aids in the control of both commensal and pathogenic microbes. The results show that the two translational products of the TSLP gene have a different expression and different biological properties, and emphasize the importance of analyzing the two TSLP isoforms separately.

A thymic stromal lymphopoietin–responsive dendritic cell subset mediates allergic responses in the upper airway mucosa

BACKGROUND Thymic stromal lymphopoietin (TSLP) controls allergic TH2 inflammatory responses through induction of distinct activation programs in dendritic cells (DCs). However, knowledge about TSLP receptor expression and functional consequences of receptor activation by DCs residing in the human respiratory tract is limited. OBJECTIVE We wanted to identify TSLP-responding DC populations in the human upper airway mucosa and assess the TSLP-mediated effects on such DCs in allergic airway responses. RESULTS We found that the TSLP receptor was constitutively and preferentially expressed by myeloid CD1c(+) DCs in the human airway mucosa and that the density of this DC subset in nasal mucosa increased significantly after in vivo allergen challenge of patients with allergic rhinitis. In vitro, TSLP strongly enhanced the capacity of CD1c(+) DCs to activate allergen-specific memory CD4(+) T cells. Moreover, TSLP rapidly induced CCR7 expression on CD1c(+) DCs. However, TH2 cytokines attenuated TSLP-mediated CCR7 induction, thus inhibiting the TSLP-induced DC migration potential to the draining lymph nodes. CONCLUSION Our results suggest that TSLP-mediated activation of human nasal mucosal CD1c(+) DCs triggers CCR7-dependent migration to the draining lymph nodes and enhances their capacity to initiate TH2 responses. However, the observation that TH2 cytokines abrogate the induction of CCR7 implies that during a TH2-mediated inflammatory reaction, TLSP-activated CD1c(+) DCs are retained in the inflamed tissue to further exacerbate local inflammation by activating local antigen-specific memory TH2 cells.