Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.

Dendritic cells (DC) are the most efficient APC for T cells. The clinical use of DC as vectors for anti-tumor and infectious disease immunotherapy has been limited by their trace levels and accessibility in normal tissue and terminal state of differentiation. In the present study, daily injection of human Flt3 ligand (Flt3L) into mice results in a dramatic numerical increase in cells co-expressing the characteristic DC markers-class II MHC, CD11c, DEC205, and CD86. In contrast, in mice treated with either GM-CSF, GM-CSF plus IL-4, c-kit ligand (c-kitL), or G-CSF, class II+ CD11c+ cells were not significantly increased. Five distinct DC subpopulations were identified in the spleen of Flt3L-treated mice using CD8 alpha and CD11b expression. These cells exhibited veiled and dendritic processes and were as efficient as rare, mature DC isolated from the spleens of untreated mice at presenting allo-Ag or soluble Ag to T cells, or in priming an Ag-specific T cell response in vivo. Dramatic numerical increases in DC were detected in the bone marrow, gastro-intestinal lymphoid tissue (GALT), liver, lymph nodes, lung, peripheral blood, peritoneal cavity, spleen, and thymus. These results suggest that Flt3L could be used to expand the numbers of functionally mature DC in vivo for use in clinical immunotherapy.

Experimental autoimmune encephalomyelitis is exacerbated by IL-1 alpha and suppressed by soluble IL-1 receptor.

To assess the role of IL-1 in the development of experimental autoimmune encephalomyelitis (EAE), the effects of in vivo treatment with IL-1 alpha or an IL-1 antagonist on the clinical course of EAE were evaluated. First, Lewis rats were immunized with guinea pig myelin in CFA and treated for 19 consecutive days with i.p. injections of recombinant human IL-1 alpha. Clinical signs of paralysis in the IL-1 alpha-treated groups were of longer duration and of greater severity compared to placebo injected controls. In addition, more weight loss was observed in the IL-1 alpha-treated groups compared to controls. This enhanced weight loss was not due to IL-1 alpha injections alone as CFA-treated rats injected with IL-1 alpha did not lose weight when compared to placebo injected, CFA-treated controls. Second, soluble mouse rIL-1R (sIL-1R), which binds both IL-1 alpha and IL-1 beta, was given as an IL-1 antagonist. Treatment of guinea pig myelin/CFA immunized rats with sIL-1R for 13 consecutive days significantly delayed the onset of EAE, reduced the severity of paralysis and weight loss, and shortened the duration of disease. Treatment with sIL-1R was most effective in reducing EAE if administered for 15 consecutive days immediately after immunization. Shortened 5-day treatment regimens spanning days 1 to 5, days 6 to 10, or days 11 to 15 after immunization were less effective in reducing EAE. These data suggest that IL-1 may initiate or promote inflammation within the central nervous system. In addition, specifically blocking the biological activity of IL-1 in vivo by soluble receptors may prove beneficial for the treatment of autoimmune or inflammatory diseases.

Human cutaneous allergic late-phase response is inhibited by soluble IL-1 receptor.

The late-phase allergic reaction (LPR) occurs 4 to 8 h after allergen exposure and probably causes the symptoms of chronic allergic disease. To determine the effects of soluble IL-1 receptor on the cutaneous LPR, we performed a prospective, randomized, double-blind, placebo-controlled study on 15 allergic subjects. Intradermal injections of allergen were placed on subjects' forearms, followed by immediate subcutaneous injections at the same site of either 1, 10, 25, 50, or 100 micrograms of rhu IL-1R to three subjects in each dosage group. Placebo was given to matched allergen-injected sites on the contralateral arm. Erythema, induration, and itching were recorded for each site. Sites were biopsied at 8 h for immunohistologic evaluations. Rhu IL-1R significantly reduced the clinical reaction at all concentrations. At 1 and 10 micrograms, measurements of LPR were significantly less (p < 0.05) than at placebo sites at several time points from 2 to 8 h. At higher concentrations, LPR was suppressed at rhu IL-1R and placebo sites, suggesting a systemic effect of rhu IL-1R. Histologic evaluation and indirect immunofluorescence for eosinophil granule major basic protein, neutrophil elastase, and mast cell tryptase showed no statistical differences between rhu IL-1R and placebo sites or among doses. IL-1 plays an important role in the generation of allergic LPR. While microgram quantities of rhu IL-1R inhibited the clinical signs and symptoms of LPR, its effects on the allergic inflammatory infiltrate are yet to be defined. In this short term trial, rhu IL-1R was neither immunogenic nor toxic.

A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function.

Dendritic cells are rare haematopoietic cells that reside in a number of organs and tissues. By capturing, processing and presenting antigens to T cells, dendritic cells are essential for immune surveillance and the regulation of specific immunity. Several members of the tumour necrosis factor receptor (TNFR) superfamily are integral to the regulation of the immune response. These structurally related proteins modulate cellular functions ranging from proliferation and differentiation to inflammation and cell survival or deaths. The functional activity of dendritic cells is greatly increased by signalling through the TNFR family member CD40. Here we report the characterization of RANK (for receptor activator of NF-kappaB), a new member of the TNFR family derived from dendritic cells, and the isolation of a RANK ligand (RANKL) by direct expression screening. RANKL augments the ability of dendritic cells to stimulate naive T-cell proliferation in a mixed lymphocyte reaction, and increases the survival of RANK+ T cells generated with interleukin-4 and transforming growth factor (TGF)-beta. Thus RANK and RANKL seem to be important regulators of interactions between T cells and dendritic cells.

Characterization and pharmacokinetic parameters of recombinant soluble interleukin-4 receptor.

The interleukin-4 receptor (IL-4R) is expressed as a 140-Kd membrane glycoprotein that binds IL-4 with high affinity. Recently, cDNA clones for the murine IL-4R have been isolated. One clone encodes an integral membrane protein, while another encodes a protein in which translation is terminated before the transmembrane region, thus producing a soluble form of the IL-4R (sIL-4R). HeLa cell clones overexpressing sIL-4R were isolated using a novel filter-overlay and 125I-IL-4 ligand binding technique. Quantitative analysis demonstrated that the kinetics and affinity of IL-4 binding to the recombinant sIL-4R were similar to the native membrane-bound IL-4R. As low doses of sIL-4R specifically inhibited IL-4-induced proliferative responses in vitro, sIL-4R biodistribution and elimination parameters were evaluated to assess the pharmacokinetic potential of sIL-4R as a therapeutic agent. Pharmacokinetic studies demonstrated that radiolabeled sIL-4R had a distribution half-life of 9 minutes and an elimination half-life of 2.3 hours following intravenous (IV) administration. When administered by intraperitoneal or subcutaneous (SC) injection, the elimination half-lives were prolonged to 4.2 hours and 6.2 hours, respectively. Although the initial blood level of sIL-4R was reduced if administered by SC injection, the bioavailability was comparable with IV administration. The main sites of sIL-4R elimination were the liver and kidney.

Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells.

The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an important role in hematopoiesis. The flt3 ligand (flt3L) is a growth factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice are treated with flt3L immature B cells, natural killer (NK) cells and dendritic cells (DC) are expanded in vivo. To further elucidate the role of flt3L in hematopoiesis, mice lacking flt3L (flt3L-/-) were generated by targeted gene disruption. Leukocyte cellularity was reduced in the bone marrow, peripheral blood, lymph nodes (LN), and spleen. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B-lymphoid progenitors were noted in the BM of flt3L-/- mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, LN, and thymus. Both myeloid-related (CD11c(++) CD8alpha(-)) and lymphoid-related (CD11c(++) CD8alpha(+)) DC numbers were affected. We conclude that flt3L has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes.