Langerhans cell migration is modulated by N-sulfated glucosamine moieties in heparin.

Dendritic cell (DC) migration into and out of tissues is important for the generation of primary immune responses to antigens encountered in tissues. In order to study the mechanisms involved in DC migration we used a skin explant system and quantitated the number of Langerhans cells (LC), which are immature precursors of DC in skin-draining lymph nodes, remaining in the epidermis in response to incubation with various biomolecules. This paper shows that LC trafficking in epidermis is a metabolically active process that is modulated by heparin, specifically by N-sulfated glucosamine moieties in heparin. This is the first demonstration of structural specificity in the biochemical requirements for DC migration in a tissue and therefore is important to understanding DC migration in general.

Avidity maturation, repertoire shift, and strain differences in antibodies to bacterial levan, a type 2 thymus-independent polysaccharide antigen.

Our previous studies of 102 mAb from mice injected with bacterial levan (BL), a beta(2-->6) linked polyfructosan with beta(2-->1) branch points (inulin determinant, In) showed that BALB/c and CBA/Ca mAb differed in VH and VL gene family usage and fine specificity. We now show that BALB/c and CBA/Ca mAb used different VHJ606 germ-line genes in response to BL: V14A in BALB/c and a previously unidentified gene in CBA/Ca. CBA/Ca mice were found to lack the BALB/c V14A gene. Also, we have compared the responses to one (primary, 1 degree) or two (secondary, 2 degrees) injections of polysaccharide. The secondary BALB/c anti-BL panel has been expanded to a total of 22 mAb, and we report here the isotype, fine specificity, and VH/VL usage of the new mAb. Eight of nine primary BALB/c In-binding mAb were germ-line, whereas both secondary BALB/c In-binding mAb that were sequenced differed from the BALB/c germ-line gene V14A. Germ-line primary mAb were low avidity whereas all five secondary mAb and the one non-germ-line primary were high avidity. There was also a repertoire shift from approximately 90% VHJ606/V kappa 11 in primary mAb to only 50% in secondary mAb (p = 0.002). The data presented provide evidence that avidity maturation and repertoire shifts, features usually associated with a memory response to thymus-dependent Ags, also can occur in response to a second immunization with a thymus-independent type 2 polysaccharide Ag.

Modulation of Ia+ Langerhans cell numbers in vivo by cultured epidermis derived supernatants and by GM-CSF.

This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.

Strain-dependent restricted VH and VL usage by anti-bacterial levan monoclonal antibodies.

The immune response to polysaccharides is highly regulated and has several distinguishing features, including restricted clonotype and isotype expression. The basis for this highly restricted response is not fully understood. To address these questions in a systematic manner, we have generated a panel of 102 mAb from CBA/CaHN (CBA/Ca) and BALB/cAnN (BALB/c) mice after one and two injections of bacterial levan (BL), a beta(2----6)-linked polyfructosan with beta(2----1)-linked fructose branch points (inulin determinant, In). This panel of mAb was examined for isotype, fine specificity, VH and VL region gene family usage and relationships between these parameters. After one or two injections of BL in both strains, mAb were IgM and IgG3. Fine specificity and VH/VL gene family usage differed markedly, however, between the two strains. Only 4% (2/51) of CBA/Ca mAb recognized the In determinant, whereas 77% (40/51) of BALB/c mAb recognized this epitope. In both strains, VH usage was restricted and certain families were overrepresented. In CBA/Ca mice, the overall response to BL was dominated by VHJ558 (45%, 23/51), the largest VH family, but VH36-60 (27%, 14/51) and VHJ606 (25%, 13/51) were also highly utilized and overrepresented. In BALB/c mice, the overall response to BL was dominated by VHJ606 (79%, 39/49 designated), a relatively small VH family. More importantly, after a single immunization with BL one particular VH/VL pair (VHJ606/ kappa 11) was used by 88% (36/41) of BALB/c mAb and was associated with In reactivity. In summary, BALB/c and CBA/Ca responses to BL differ in fine specificity and VH/VL usage.