The extracellular matrix component hyaluronan (HA) exists physiologically as a high m.w. polymer but is cleaved at sites of inflammation, where it will be contacted by dendritic cells (DC). To determine the effects of HA on DC, HA fragments of different size were established. Only small HA fragments of tetra- and hexasaccharide size (sHA), but not of intermediate size (m.w. 80,000–200,000) or high m.w. HA (m.w. 1,000,000–600,000) induced immunophenotypic maturation of human monocyte-derived DC (up-regulation of HLA-DR, B7-1/2, CD83, down-regulation of CD115). Likewise, only sHA increased DC production of the cytokines IL-1β, TNF-α, and IL-12 as well as their allostimulatory capacity. These effects were highly specific for sHA, because they were not induced by other glycosaminoglycans such as chondroitin sulfate or heparan sulfate or their fragmentation products. Interestingly, sHA-induced DC maturation does not involve the HA receptors CD44 or the receptor for hyaluronan-mediated motility, because DC from CD44-deficient mice and wild-type mice both responded similarly to sHA stimulation, whereas the receptor for hyaluronan-mediated motility is not detectable in DC. However, TNF-α is an essential mediator of sHA-induced DC maturation as shown by blocking studies with a soluble TNFR1. These findings suggest that during inflammation, interaction of DC with small HA fragments induce DC maturation.

IntroductionOsteopontin (OPN) is a secreted phosphoprotein that contains an integrin-binding arginine-glycine-aspartate sequence (RGD) motif. OPN has proinflammatory cytokine and chemokine functions in cell-mediated immunity. [1][2][3][4] A number of studies have demonstrated that OPN critically contributes to the development of T helper 1 (Th1)-mediated immunity and disease. 5,6 Among other reports, OPN-deficient mice develop disseminated infection and have a delayed ability to clear disease when infected with Mycobacterium bovis (BCG). 7 In murine models of autoimmune encephalomyelitis, a model of human multiple sclerosis that critically depends upon the balance of Th-shaping cytokines such as interleukin-10 (IL-10) and IL-12, OPN-deficient mice develop milder disease. 8,9 Corneal infection of OPN-deficient mice with herpes simplex virus 1 (HSV-1) is followed by a reduced delayedtype hypersensitivity to HSV. 6 We have shown that OPN-deficient mice have an impaired allergic contact hypersensitivity (CHS) response against trinitrochlorobenzene, which is accompanied by a reduced ability to attract dendritic cells (DCs) from the periphery into draining lymph nodes. 10 DCs are the most potent antigen-presenting cells (APCs). Their function and polarizing capacities are decisive for the outcome of Th-mediated immunity. [11][12][13] Although, it is known that OPN plays a central role for the initiation of Th1-mediated immune responses, it is unknown whether it is involved in DC instruction to induce Th1-mediated responses. In their immature state, DCs are situated in peripheral nonlymphoid tissues. 14,15 For example, Langerhans cells (LCs) are located in the epidermis. Upon stimulation, LCs/DCs undergo a maturation process resulting in the downregulation of their antigen uptake and processing capacity; upregulation of major histocompatibility complex (MHC) II and costimulatory molecules; and a switch in their expression pattern of chemokines and adhesion molecules. 14,15 As a consequence of this reprogramming, DCs migrate into lymphoid organs. 16,17 In the T-cell zone of lymph nodes, they function as APCs, which prime naive antigen-specific T cells and drive their differentiation toward Th1, Th2, or regulatory T cells. [11][12][13] The initial activation stimulus in concert with tissue environmental factors encountered by migrating DCs instruct DCs to polarize toward a phenotype that initiates Th1, Th2, or regulatory T effector cells. 11,12,18,19 Numerous viral and microbial factors, among them lipopolysaccharide (LPS), Staphylococcus aureus Cowan strain I, bacterial DNA and dsRNA, [20][21][22][23][24] and Pertussis toxin in the context of IL-1␤ and tumor necrosis factor ␣ (TNF-␣), 25 have been described as IL-12-and Th1-prompting factors. In contrast, substances that increase intracellular cyclic adenosine monophosphate (cAMP), such as cholera toxin 26 For personal use only. on May 9, 2018. by guest www.bloodjournal.org From Incomplete knowledge exists regarding tissue factors that are encountered by DCs on their w...

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and αv integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

Dendritic cells (DC) have an increasingly important role in vaccination therapy; therefore, this study sought to determine the migratory capacity and immunogenic function of murine bone-marrow (BM)-derived DC following subcutaneous (s.c.) and intravenous (i.v.) injection in vivo. DC were enriched from BM cultures using metrizamide. Following centrifugation, the low-buoyant density cells, referred to throughout as DC, were CD11c(high), Iab(high), B7-1(high) and B7-2(high) and potently activated alloreactive T cells in mixed lymphocyte reactions (MLR). In contrast, the high-density cells expressed low levels of the above markers, comprised mostly of granulocytes based on GR1 expression, and were poor stimulators in MLR. Following s.c. injection of fluorescently labelled cells into syngeneic recipient mice, DC but not granulocytes migrated to the T-dependent areas of draining lymph nodes (LN). DC numbers in LN were quantified by flow-cytometric analysis, on 1, 2, 3, 5 and 7 days following DC transfer. Peak numbers of around 90 DC per draining LN were found at 2 days. There was very little migration of DC to non-draining LN, thymus or spleen at any of the time-points studied. In contrast, following i.v. injection, DC accumulated mainly in the spleen, liver and lungs of recipient mice but were largely absent from peripheral LN and thymus. The ability of DC to induce T-cell-mediated immune responses was examined using trinitrobenzenesulphate (TNBS)-derivatized DC (TNBS-DC) to sensitize for contact hypersensitivity responses (CHS) in naive syngeneic recipients. Following s.c. injection, as few as 105 TNBS-DC, but not TNBS-granulocytes, sensitized for CHS responses. However, the same number of TNBS-DC failed to induce CHS following i.v. injection. In summary, this study provides new and quantitative data on the organ specific migration of murine BM-derived DC following s.c. and i.v. injection. The demonstration that the route of DC administration determines the potency of CHS induction, strongly suggests that the route of immunization should be considered in the design of vaccine protocols using DC.