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The mechanisms controlling the exit of lymphocytes from tissues via lymphatics are practically unknown. We have now identified a 270-300-kDa molecule designated common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) on human lymphatic endothelium and high endothelial venules. We show that it mediates binding of lymphocytes both to high endothelial venules and to lymphatic vessels. Moreover, blocking of the function of CLEVER-1 results in significant reduction of lymphocyte traffic in vivo. Notably, CLEVER-1 is also an inducible vascular adhesion molecule for other classes of leukocytes at sites of inflammation in peripheral tissues. These findings suggest that CLEVER-1 is involved in regulation of lymphocyte recirculation and migration of leukocytes to sites of inflammation and is a potential new target to control inflammation.

Knowledge of the histological distribution of leucocytes and adhesion molecules in the human genital tract is scarce although local immunity in this region is important. Using immunohistochemical methods, we here describe the organization of CD3+, CD8+ and CD4+ T cells, CD19+ B cells, CD38+ plasma cells, major histocompatibility complex (MHC) class II+ antigen-presenting cells and CD14+ monocytes, as well as the expression of endothelial addressins in normal human ecto-cervical and vaginal mucosa. T cells were clustered in a distinct band beneath the epithelium and were also dispersed in the epithelium and the lamina propria, whereas CD38+ plasma cells were present only in the lamina propria. MHC class II+ cells were numerous in the lamina propria and in the epithelium, where they morphologically resembled dendritic cells. Lymphoid aggregates containing CD19+ and CD20+ B cells as well as CD3+, CD4+ and CD8+ cells were also found in the cervix. The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was not expressed on the vascular endothelium in the cervical or vaginal mucosa. In contrast, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion protein-1 (VAP-1) and P-selectin were expressed in all tissue samples, and vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were found in four of seven samples. We conclude that the distribution of leucocytes and adhesion molecules is very similar in the ecto-cervical and the vaginal mucosa and that the regulation of lymphocyte homing to the genital tract is different from that seen in the intestine. Our results also clearly suggest that the leucocytes are not randomly scattered in the tissue but organized in a distinct pattern.

Helicobacter pylori infection induces chronic inflammation in the gastric mucosa with a marked increase in the number of lymphoid follicles consisting of infiltrating B and T cells, neutrophils, dendritic cells (DC) and macrophages. It has been suggested that an accumulation of mature DC in the tissue, resulting from a failure of DC to migrate to lymph nodes, may contribute to this chronic inflammation. Migration of DC to lymph nodes is regulated by chemokine receptor CCR7, expressed on mature DC, and the CCR7 ligands CCL19 and CCL21. In this study we analysed the maturation, in vitro migration and cytokine production of human DC after stimulation with live H. pylori. For comparison, DC responses to non-pathogenic Escherichia coli bacteria were also evaluated. Stimulation with H. pylori induced maturation of DC, i.e. up-regulation of the chemokine receptors CCR7 and CXCR4 and the maturation markers HLA-DR, CD80 and CD86. The H. pylori-stimulated DC also induced CD4+ T-cell proliferation. DC stimulated with H. pylori secreted significantly more interleukin (IL)-12 compared to DC stimulated with E. coli, while E. coli-stimulated DC secreted more IL-10. Despite low surface expression of CCR7 protein following stimulation with H. pylori compared to E. coli, the DC migrated equally well towards CCL19 after stimulation with both bacteria. Thus, we could not detect any failure in the migration of H. pylori stimulated DC in vitro that may contribute to chronic gastritis in vivo, and our results suggest that H. pylori induces maturation and migration of DC to lymph nodes where they promote T cell responses.

Homing of leukocytes to various tissues is dependent on the interaction between homing receptors on leukocytes and their ligands, addressins, on endothelial cells. Mucosal immunization results in homing of antigen-specific lymphocytes back to the mucosa where they first encountered the antigen. However, it is unknown whether this homing of antigen-specific cells is mediated by an altered endothelial addressin expression after vaccination. Using different immunization routes with an oral cholera vaccine, we show that the endothelial expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is increased in the gastric and upper small intestinal mucosae after immunization through various local routes in the upper gastrointestinal tract. In contrast, rectal immunization did not influence the levels of MAdCAM-1 in the gastric or duodenal mucosa. Furthermore, we show that MAdCAM-1 can be induced on human endothelial cells by tumor necrosis factor alpha (TNF-α) and gamma interferon. The vaccine component cholera toxin B subunit (CTB) increased MAdCAM-1 expression on endothelial cells in cultured human gastric explants, an effect that seemed to be mediated by TNF-α. In conclusion, MAdCAM-1 expression is increased in the upper gastrointestinal tract after local immunizations with a vaccine containing CTB. This strongly suggests the involvement of MAdCAM-1 in the preferential homing of mucosal lymphocytes to their original site of activation.

Determination of optimal routes to induce mucosal immune responses locally in the stomach and duodenum are important steps in the development of vaccines against Helicobacter pylori infection. In this study, we immunized H. pylori-infected individuals either nasally or rectally with a model antigen, i.e. cholera toxin B subunit, and compared the immune responses after these routes with the responses after oral or intrajejunal vaccination. Specific antibody levels in serum as well as specific antibody levels and antibody-secreting cells in biopsies from antrum and duodenum were determined by ELISA and ELISPOT methods. In contrast to oral vaccination, nasal and rectal vaccination did not induce significant increases in specific antibody-secreting cells either in the antrum or duodenum. Furthermore, when analyzing the antibody levels in saponin extracted biopsies, intrajejunal vaccination was superior to both nasal and rectal vaccination in inducing antigen-specific IgA levels in the stomach. We conclude that oral vaccination is the optimal route for induction of antigen-specific IgA antibody responses in the stomach and duodenum of humans, while nasal or rectal vaccination is less suitable for this purpose.

Sexually transmitted diseases are a major health problem worldwide, but there is still a lack of knowledge about how to induce an optimal immune response in the genital tract of humans. In this study we vaccinated 21 volunteers nasally or vaginally with the model mucosal antigen cholera toxin B subunit and determined the level of specific immunoglobulin A (IgA) and IgG antibodies in vaginal and cervical secretions as well as in serum. To assess the hormonal influence on the induction of antibody responses after vaginal vaccination, we administered the vaccine either independently of the stage in the menstrual cycle or on days 10 and 24 in the cycle in different groups of subjects. Vaginal and nasal vaccinations both resulted in significant IgA and IgG anti-cholera toxin B subunit responses in serum in the majority of the volunteers in the various vaccination groups. Only vaginal vaccination given on days 10 and 24 in the cycle induced strong specific antibody responses in the cervix with 58-fold IgA and 16-fold IgG increases. In contrast, modest responses were seen after nasal vaccination and in the other vaginally vaccinated group. Nasal vaccination was superior in inducing a specific IgA response in vaginal secretions, giving a 35-fold increase, while vaginal vaccination induced only a 5-fold IgA increase. We conclude that a combination of nasal and vaginal vaccination might be the best vaccination strategy for inducing protective antibody responses in both cervical and vaginal secretions, provided that the vaginal vaccination is given on optimal time points in the cycle.

Continuous lymphocyte recirculation between blood and lymphoid tissues forms a basis for the function of the immune system. Lymphocyte entrance from the blood into the tissues has been thoroughly characterized, but mechanisms controlling lymphocyte exit from the lymphoid tissues via efferent lymphatics have remained virtually unknown. In this work we have identified mannose receptor (MR) on human lymphatic endothelium and demonstrate its involvement in binding of lymphocytes to lymphatic vessels. We also show that the binding requires L-selectin, and L-selectin and MR form a receptor–ligand pair. On the other hand, L-selectin binds to peripheral lymph node addressins (PNAds) on high endothelial venules (HEVs) that are sites where lymphocytes enter the lymphatic organs. Interestingly, MR is absent from HEVs and PNAds from lymphatic endothelium. Thus, lymphocyte L-selectin uses distinct ligand molecules to mediate binding at sites of lymphocyte entrance and exit within lymph nodes. Taken together, interaction between L-selectin and MR is the first molecularly defined mechanism mediating lymphocyte binding to lymphatic endothelium.

Although nasal vaccination has emerged as an interesting alternative to systemic or oral vaccination, knowledge is scarce about the immune responses after such immunization in humans. In the present study, we have compared the kinetics and organ distribution of the antibody responses after nasal and oral vaccination. We immunized female volunteers nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum and nasal and vaginal secretions, as well as the number of circulating antibody-secreting cells, before immunization and 1, 2, 3, 6, and 26 weeks thereafter. Nasal vaccination induced 9-fold CTB-specific immunoglobulin A (IgA) and 56-fold specific IgG antibody increases in nasal secretions, whereas no significant IgA increase was seen after oral vaccination. Both oral and nasal vaccination resulted in 5- to 6-fold CTB-specific IgA and 20- to 30-fold specific IgG increases in vaginal secretions. Strong serum responses to CTB were also induced by both routes of vaccination. A notable difference between nasal and oral vaccination was that the nasal route elicited a specific antibody response with a later onset but of much longer duration than did the oral route. We conclude from this study that the nasal route is superior to the oral route for administering at least nonliving vaccines against infections in the upper respiratory tract, whereas either oral or nasal vaccination might be used for eliciting antibody responses in the female genital tract.

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.

Forty-five volunteers were vaccinated twice intranasally with 10, 100, or 1,000 microg of cholera toxin B subunit (CTB). Blood and nasal and vaginal secretions were collected before and 1 week after the second vaccination from all volunteers, and the specific and total immunoglobulin A (IgA) and IgG titers were determined by enzyme-linked immunosorbent assay. Samples were also taken 6 months (n = 16) and 1 year (n = 14) after the vaccination. The 10- and 100-microg doses were well tolerated by the volunteers, but the 1,000-microg dose induced increased secretions from the nose and repetitive sneezings for several hours. The CTB-specific serum IgA and IgG increased 21- and 7-fold, respectively, 1 week after vaccination with the medium dose and increased 61- and 37-fold, respectively, after the high dose. In nasal secretions the specific IgA and IgG increased 2- and 6-fold after the medium dose and 2- and 20-fold after the high dose, respectively. In vaginal secretions the specific IgA and IgG increased 3- and 5-fold after the medium dose and 56- and 74-fold after the high dose, respectively. The lowest dose did not induce any significant antibody titer increases in serum or in secretions. The specific IgA and IgG levels in secretions were still elevated after 6 months but were decreasing 1 year after the vaccination. These results show that intranasal vaccination of humans with CTB induces strong systemic and mucosal antibody responses and suggest that CTB may be used as a carrier for antigens that induce protective immunity against systemic as well as respiratory and genital infections.