Jane Guo

APRIL and TALL-1 and receptors BCMA and TACI: system for regulating humoralimmunity

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-1 (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-1 signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL–TALL-1 and BCMA-TACI form a two ligands–two receptors pathway involved in stimulation of B and T cell function.

IL-18-Binding Protein Protects Against Lipopolysaccharide- Induced Lethality and Prevents the Development of Fas/Fas Ligand-Mediated Models of Liver Disease in Mice

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (KD 0.3–5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-γ production by KG1 cells (EC50 = 0.3 μg/ml). In mice challenged with an LD90 of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-γ production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-γ production induced by LPS (5 mg/kg) with ED50 of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-γ production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-γ and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1α and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-γ and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.

TNF-induced enterocyte apoptosis in mice is mediated by the TNF receptor 1 and does not require p53.

Injection of recombinant mouse TNF into mice is known to induce a shrinkage of the duodenal villi, which becomes evident 30 – 90 min later and is associated with a detachment of enterocytes in the lumen. These cells can be collected by lavage and are all apoptotic, i.e. hypodiploid as seen by flow cytometric analysis. Thus the count of detached cells was used as an evaluation of the TNF‐induced cell loss and apoptosis in the mucosa. TNF injection induced a cell loss of similar magnitude in wild‐type (+/+) or in mice lacking the TNF receptor (TNFR)2 (p75, TNFR2 −/−), while mice lacking the TNFR1 (p55, TNFR1 −/−) were completely resistant to this effect. TNF increased the expression of p53 tumor suppressor gene in the enterocytes from the crypts but not from the villi, as seen by Western blots and histochemistry. TNF increased the expression of p53 in both TNFR2 −/− and TNFR1 −/− mice. Furthermore, enterocyte cell loss was not attenuated in p53 −/− mice. The results indicate that TNF, acting on its receptor 1, induces an apoptotic detachment of the enterocytes from the tip of the villi ( i.e. the old enterocytes), while in the enterocytes from the crypts (the young enterocytes) TNF increases, via either TNFR1 or TNFR2, the expression of p53, without inducing apoptosis.

Stimulatory Effects of B7-Related Protein-1 on Cellular and Humoral Immune Responses in Mice

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-γ, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.

Heparin attenuates bleomycin but not silica-induced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis

Pulmonary fibrosis was elicited in mice or rats by the intra‐tracheal instillation of bleomycin or silica. Daily injections of heparin significantly reduced the collagen deposition in bleomycin, but not in silica, injected mice, as evaluated by the lung hydroxyproline content on day 15 after instillation. Heparin also reduced the bleomycin‐induced morbidity and mortality. Study of the broncho‐alveolar lavage fluid (BAL) detected no significant change in the number of leucocytes or the amount of protein in heparin treated mice. Histologies of bleomycin instilled mice suggested that heparin did reduce the alveolar remodelling but not the alveolitis, evidenced by leucocytic infiltration. As detected by electron microscopy (EM), bleomycin increased the number of leucocytes and platelets within the alveolar capillaries but this was not significantly reduced by heparin. The phenotype of the interstitial cell involved in these two types of pulmonary fibrosis was investigated by immunohistochemistry and EM. While in bleomycin injected animals the interstitial cells had the phenotype of an actin (α‐actin in the rat) and lipid containing interstitial cell, with a poorly developed RE, in silica injected animals in contrast, the interstitial cells were without cytoplasmic actin or lipid but with a markedly developed endoplasmic reticulum (ER). Thus bleomycin and silica induced the growth of two different types of interstitial cells, the myofibroblast and the regular fibroblast, which might be a reason why heparin selectively inhibits bleomycin but not silica‐induced fibrosis.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

Stimulation of thrombocytopoiesis decreases platelet β2 but not β1 or β3 integrins

The expression of CD29, CD61, CD18 and CD11a on platelets was examined by flow cytometry in mice treated with leukaemia inhibitory factor (LIF) or megakaryocyte growth and development factor (PEG‐rHuMGDF or mpl‐ligand). Treatment for 7–14 d with PEG‐rHuMGDF or LIF increased the number of platelets in peripheral blood from 0.9 up to <2.0×106/μl. These treatments decreased the expression of CD11a and CD18, whereas that of CD29 or CD61 was not markedly changed. Study after various doses or times of PEG‐rHuMGDF administration indicated that a decrease of CD18 expression occurred when platelet counts started to rise. Platelet RNA content was increased in mice treated with PEG‐rHuMGDF but double staining indicated that expression of CD18 was not correlated with RNA content. To evaluate integrin expression as a function of time in circulation, platelets were biotinylated in vivo. In normal or PEG‐rHuMGDF‐treated mice, the expression of CD29 or CD61 did not change, whereas that of CD18 decreased significantly as a function of time in circulation. These findings indicate, firstly, that stimulation of thrombocytopoiesis leads to the release of platelets with a low content of β2 integrin and, secondly, that this integrin is also selectively lost while in the circulation.