Normal Mouse Spleen Research Articles

A factor isolated from Taenia solium metacestodes stimulates T lymphocytes to proliferate and produce gamma interferon.

A metacestode factor (MF) isolated from live metacestodes of Taenia solium suppresses humoral and cellular responses, and inhibits the inflammatory reaction around metacestodes implanted subcutaneously in mice. When this MF is digested with RNase (dMF), it loses the suppressive capacity, but acquires T-cell stimulant ability. By filtering MF through a Bio-gel P6 column, two components were separated. The first (F1) was suppressive. while the second (F2) stimulated T cells to proliferate. In these experiments, F2 or dMF was used with mouse spleen cells in stimulation assays in vitro. Spleen cells from mice treated with F2 or dMF were also stimulated with concanavalin A (Con-A) ex vivo. Flow cytometry analyses were performed to estimate cell proliferation, intracellular cytokine production. and restoration of CD4 cells. Spleen lymphocytes from mice previously treated with F2 or dMF and then stimulated with Con-A ex vivo exhibited a significant increase in cell proliferation and gamma interferon production by CD4+ (P<0.05) and CD8+ cells. These effects were concentration-dependent and inversely correlated with the amount of dMF or F2. Similar results were observed in normal mouse spleen T cells incubated with F2 or dMF and Con-A in vitro. Finally, dMF induced a significant restoration of CD4-cells in mice depleted of these cells.

Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration.

CD25(+)CD4(+) T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25(+)CD4(+) to CD25(-)CD4(+) cells with an increase of CD25(+)CD4(+) T cells accompanied by a decrease of CD25(-)CD4(+) T cells. The relative increase in CD25(+)CD4(+) T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25(+)CD4(+) T cells and there was an increase in the percentage of CD25(+)CD4(+) T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25(-)CD4(+) cells, CD25(+)CD4(+) cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-beta(1) following anti-CD3 stimulation. CD25(+)CD4(+) cells from fed mice suppressed the proliferation of CD25(-)CD4(+) T cells in vitro more potently than CD25(+)CD4(+) T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-beta soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25(+)CD4(+) cells from unfed mice secreted IFN-gamma, whereas CD25(+)CD4(+) cells from fed mice did not. Adoptive transfer of CD25(+)CD4(+) T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25(+)CD4(+) regulatory T cells and suggest that they may be involved in oral tolerance.

GLYCYRRHIZIN RESTORES THE IMPAIRED IL-12 PRODUCTION IN THERMALLY INJURED MICE

Mice 6 days after thermal injury (TI-mice) did not respond to lipopolysaccharide (LPS) stimulation for production of serum interleukin 12 (IL-12; 2h after LPS stimulation, <20pg/ml in TI-mice; 1091±162pg/ml in normal mice). However, 2h after LPS stimulation, 1456±118pg/ml of IL-12 were demonstrated in sera of TI-mice previously treated with a 10mg/kg i.p. dose of glycyrrhizin (GR). IL-12 was not induced by LPS in sera of normal mice inoculated with burn-associated type 2 T cells (IL-4/IL-10-producing CD8+CD11b+TCRγ/δ+T cells isolated from spleens of TI-mice). However, IL-12 production was induced by LPS in sera of these mice previously treated with GR or a mixture of monoclonal antibodies (mAbs) for type 2 cytokines. Also, IL-12 production was induced by LPS in TI-mice inoculated with CD4+T cells from spleens of GR-treated normal mice (GR-CD4+T cells, 5×106cells/mouse). Since GR-CD4+T cells have been shown to be antagonistic cells against production of type 2 cytokines by burn-associated type 2 T cells, these results indicate that IL-12 unresponsiveness shown in TI-mice is recovered by GR through the regulation of burn-associated type 2 T cell responses.

The fate of adoptively transferred quiescent encephalitogenic T cells in normal and antigen-tolerized mice

Adoptive transfer of quiescent encephalitogenic T cells to normal syngeneic recipients was without clinical effect. RT-PCR was used to assess localization of an adoptively transferred quiescent encephalitogenic T cell clone in normal and antigen-unresponsive mice prior to or after challenge with neuroantigen/CFA. The T cell clone was not detectable in lymphoid tissues prior to challenge with neuroantigen; however, following challenge, the clone was found in the spleen, lymph nodes and spinal cord of both normal and antigen-tolerized mice. The latter animals remained clinically normal. Non-activated encephalitogenic T cells transferred to wild-type recipients pretreated i.p. with neuroantigen/IFA were rendered unresponsive. Transfer of the same T cells to α/β T cell-deficient mice pretreated with neuroantigen/IFA resulted in spontaneous disease indicating that an intact α/β T cell system was required for development of the unresponsive state.

E-Selectin Expression in a Murine Model of Chronic Colitis

The objective of this study was to quantify E-selectin surface expression in the colon as well as other tissues in a CD4+ T-cell model of chronic colitis in mice using the newly developed dual radiolabel monoclonal antibody technique. Male SCID mice were reconstituted with either 5 × 105 CD4+ CD45RBlow or CD45RBhigh T-cells isolated from normal CB-17 donor mouse spleens and subsequently monitored for clinical signs of colitis. We found that animals injected with CD45RBhigh but not CD45RBlow T-cells nor PBS developed colitis at 6–8 weeks following reconstitution as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of E-selectin compared to SCID mice injected with PBS or reconstituted with CD45RBlow T-cells, both of which did not develop colitis. We also observed significant increases in E-selectin expression in cecum, small intestine, mesentery, and liver of colitic mice. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T-cells induces chronic colitis and demonstrate that this chronic colitis is associated with enhanced expression of an endothelial cell-specific adhesion molecule. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T-cells enhances E-selectin expression in a variety of tissues distant from the site of active inflammation.

Structure and function of the murine chemokine receptor CXCR3

The gene encoding the murine homologue of human CXCR3 exists in a single copy consisting of two exons with an intron interrupting the coding sequence between nucleotides 10 and 11. The deduced amino acid sequence is 86 % identical to the predicted human sequence. Murine CXCR3 mRNA is detectable in bone marrow cells cultured in the presence of IL-2 but not unstimulated cells. It is also detectable at low abundance in normal mouse spleen, lymph node, mammary gland, and thymus. Transfection of murine CXCR3 in murine pre-B lymphocyte line (CXCR3 / L1.2) conferred binding of the ligands IP10, ITAC and Mig with KD's of 1.35 ± 0.56, 1.41 ± 0.20, and 11.65 ± 0.90 nM, respectively. Lower affinity binding was observed for several beta or CC chemokines (eotaxin, MCP-3, MIP3α and SLC / 6Ckine / Exodus 2). ITAC, IP10 and Mig induced chemotaxis with an order of potency ITAC > IP10 = Mig. The chemokines also increased intracellular calcium concentration and were variably desensitized to repeated agonist stimulation. The hierarchy for cross- desensitization was ITAC > Mig > IP10. Thus, while Mig, ITAC and IP10 all act on the same receptor for binding and agonist stimulation, they may interact with different receptor conformational isoforms to produce divergent responses.

Structure and function of the murine chemokine receptor CXCR3.

The gene encoding the murine homologue of human CXCR3 exists in a single copy consisting of two exons with an intron interrupting the coding sequence between nucleotides 10 and 11. The deduced amino acid sequence is 86% identical to the predicted human sequence. Murine CXCR3 mRNA is detectable in bone marrow cells cultured in the presence of IL-2 but not unstimulated cells. It is also detectable at low abundance in normal mouse spleen, lymph node, mammary gland, and thymus. Transfection of murine CXCR3 in murine pre-B lymphocyte line (CXCR3++/L1.2) conferred binding of the ligands IP10, ITAC and Mig with K(D)'s of 1.35 +/- 0.56, 1.41 +/- 0.20, and 11.65 +/- 0.90 nM, respectively. Lower affinity binding was observed for several beta or CC chemokines (eotaxin, MCP-3, MIP3alpha and SLC/6Ckine/Exodus 2). ITAC, IP10 and Mig induced chemotaxis with an order of potency ITAC > IP10 = Mig. The chemokines also increased intracellular calcium concentration and were variably desensitized to repeated agonist stimulation. The hierarchy for cross- desensitization was ITAC > Mig > IP10. Thus, while Mig, ITAC and IP10 all act on the same receptor for binding and agonist stimulation, they may interact with different receptor conformational isoforms to produce divergent responses.

Ethanol decreases host resistance to pulmonary metastases in a mouse model: role of natural killer cells and the ethanol-induced stress response.

The present study was done to delineate cause-effect relationships between the ethanol (EtOH)-induced stress response, natural-killer (NK)-cell activity, and resistance to experimental metastases of B16F10 melanoma cells in mice. Increased numbers of metastatic nodules were noted in the lungs of mice treated with dosages of EtOH that produce peak blood levels of 0.25-0.4%. EtOH caused only a minor depletion of NK cells or NK-cell activity from the spleen or lungs of normal or B16F10-challenged mice. However, in earlier studies we have shown consistent, significant decreases in NK-cell activity (approx. 50%) in spleen preparations from EtOH-treated mice. Depletion of NK cells by a monoclonal antibody increased the number of B16F10 nodules in the lungs, confirming an important role for NK cells for resistance to B16F10 metastases. Treatment of NK-cell-depleted mice with EtOH caused no further decrease in resistance to B16F10 cells, indicating that suppression of NK-cell activity is the major mechanism by which EtOH suppresses resistance to B16F10 metastases. Adrenalectomy or a glucocorticoid antagonist partially prevented EtOH-induced increases in the number of metastatic nodules in the lungs. Administration of exogenous corticosterone increased the number of B16F10 nodules to an extent similar to that caused by EtOH. These results indicate a role for the EtOH-induced stress response in decreasing resistance to B16F10 metastases. EtOH-induced decreases in resistance to cancer have also been reported in rats. The findings of the present study support the possibility that this is a generalized phenomenon, which could occur in humans.

Ethanol decreases host resistance to pulmonary metastases in a mouse model: Role of natural killer cells and the ethanol-induced stress response

The present study was done to delineate cause-effect relationships between the ethanol (EtOH)-induced stress response, natural-killer (NK)-cell activity, and resistance to experimental metastases of B16F10 melanoma cells in mice. Increased numbers of metastatic nodules were noted in the lungs of mice treated with dosages of EtOH that produce peak blood levels of 0.25–0.4%. EtOH caused only a minor depletion of NK cells or NK-cell activity from the spleen or lungs of normal or B16F10-challenged mice. However, in earlier studies we have shown consistent, significant decreases in NK-cell activity (approx. 50%) in spleen preparations from EtOH-treated mice. Depletion of NK cells by a monoclonal antibody increased the number of B16F10 nodules in the lungs, confirming an important role for NK cells for resistance to B16F10 metastases. Treatment of NK-cell-depleted mice with EtOH caused no further decrease in resistance to B16F10 cells, indicating that suppression of NK-cell activity is the major mechanism by which EtOH suppresses resistance to B16F10 metastases. Adrenalectomy or a glucocorticoid antagonist partially prevented EtOH-induced increases in the number of metastatic nodules in the lungs. Administration of exogenous corticosterone increased the number of B16F10 nodules to an extent similar to that caused by EtOH. These results indicate a role for the EtOH-induced stress response in decreasing resistance to B16F10 metastases. EtOH-induced decreases in resistance to cancer have also been reported in rats. The findings of the present study support the possibility that this is a generalized phenomenon, which could occur in humans. Int. J. Cancer 82:886–892, 1999. © 1999 Wiley-Liss, Inc.

Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity.

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.

Target cells for an immunosuppressive cytokine, glycosylation-inhibiting factor.

Receptors for bioactive glycosylation-inhibiting factor (GIF) were demonstrated using a bioactive mutant of recombinant human (rh) GIF, which is comparable to the suppressor T (Ts) cell-derived bioactive GIF in its affinity for the receptors on helper T (Th) hybridoma cells. Both naive T and B cells in normal mouse spleen lacked GIF receptors. However, presentation of specific antigen to naive T cells resulted in the expression of the receptors on activated T cells. Furthermore, activation of small resting B cells with F(ab')2 fragments of anti-mouse IgM plus IL-4, lipopolysaccharide (LPS) plus IL-4 or LPS plus dextran sulfate induced the expression of the receptors within 48 h of B cell stimulation. It was also found that NK T cells freshly isolated from mouse spleen, but not conventional NK cells, expressed receptors for GIF. CD4(+) and CD4(-) subpopulations of NK T cells showed a similar binding capability. Mature dendritic cells derived from bone marrow did not bear the receptors. The dissociation constant (Kd) of the interaction between the bioactive rhGIF mutant and the high-affinity receptors was 10-100 pM, whereas inactive wild-type rhGIF failed to bind to the receptors. A bioactive derivative of rhGIF suppressed both IgG1 and IgE synthesis by purified B cells activated by LPS and IL-4, indicating that the binding of bioactive GIF to its receptors on activated B cells results in suppression of their differentiation.

Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.

Donor Treatment with Flt3 Ligand Augments Microchimerism in Tacrolimus-Immunosuppressed Allogeneic Bone Marrow Recipients

162 Flt3 ligand (FL) is a potent hematopoietic growth factor shown to mobilize stem and progenitor cells, and to selectively and strikingly augment dendritic cells in vivo. The Aim was to examine the influence of FL-mobilized donor bone marrow (BM) on microchimerism and anti-donor immune reactivity in transiently immunosuppressed recipients. Methods: B10 (H2b) mice were treated with FL for 10 days (10 μg/d) before use as BM donors. Phenotypic analysis of surface antigens was performed on freshly-isolated BM cells by flow cytometry. Normal C3H (H2k) recipient mice were injected i.v. with 50 × 106 freshly-isolated FL-mobilized or normal donor BM cells. Groups also received tacrolimus immunosuppression (2 or 4 mg/kg/d, d 0-12). Microchimerism, and anti-donor immune reactivity were evaluated 15 and 30 days post-BM transplantation. Donor MHC class II+ (IAb+) cells, were quantitated in recipient lymphoid tissues (spleen and thymus) by immunohistochemical analysis. Anti-donor reactivity was evaluated by MLR and CTL assays. Results: Phenotypic analysis of BM cells revealed significantly higher numbers of Sca-1, MHC class I and II, CD8 and CD11c expressing cells in FL-treated compared to normal donors. There was no significant difference in CD40, CD80, CD86, MAC-1, GR-1 or CD4 expression. Post-transplantation, low numbers of donor cells were detected on day 15 in the spleen and thymus of normal mice given either normal or FL-mobilized donor BM cells. By day 30, these cells were no longer detected. However, addition of tacrolimus led to a substantial increase in chimerism in these groups on day 15. A two-fold increase in donor cells was seen in animals given FL-mobilized BM + tacrolimus compared to those given normal BM + tacrolimus alone. On day 30, donor cells were detected in the spleen and thymus of only the FL-mobilized BM + tacrolimus group. At both time points, maximal anti-donor MLR and CTL activities were detected in the groups given either normal or FL-mobilized BM, with minimal activity in the tacrolimus-treated groups. Conclusion: Administration of FL to donor mice induced a significant increase in the number of stem cells and DC progenitors in the BM. When injected into allogeneic recipients under cover of immunosuppression, these cells survived longer and in greater numbers when compared to normal BM cells. This suggests that FL-mobilized donor BM can augment donor microchimerism in non-cytoablated, conventionally immunosuppressed graft recipients, and may be useful as a therapeutic strategy to prolong organ allograft survival.

Trypanosoma musculi:Tracking Parasites and Circulating Lymphoid Cells in Host Mice

Albright, J. W., Mease, R. C., Lambert, C., and Albright, J. F. 1999.Trypanosoma musculi:Tracking parasites and circulating lymphoid cells in host mice.Experimental Parasitology91,185–195. Two aspects of host-parasite relationships that seem worthy of more attention are: (a) the distribution of parasites among host organs in the early course of infection, and (b) the dynamics of host lymphocyte tissue localization and recirculation during the course of infection. We have employed the derivatized aminostyrylpyridinium dye, [125I] I 2P-Di-6-ASP, to provide a relatively stable tag on both a parasite,Trypanosoma musculi, and on host mouse splenocytes, enriched B and T lymphocytes, and natural killer cells. The organ distribution of the parasites, splenocytes, and lymphocytes in recipient, host mice was tracked. RadiolabeledT. musculilocalized primarily in the liver with lesser numbers in spleen, lungs, and kidneys. Per unit wet weight, the spleen accumulated parasites most efficiently. WhenT. musculiwere inoculated intraperitoneally, most of them remained in the peritoneal space and the numbers that gained access to liver, lungs, and spleen were significantly smaller than in mice inoculated intravenously. The acquisition of parasites by the spleen (and lungs) of mice with an existingT. musculiinfection was markedly inhibited. This was true also of syngeneic splenocytes and lymphocytes. In addition, lymphocytes from infected mice were significantly less likely to take residence in the spleens of normal recipient mice and were especially unlikely to localize in the spleens of infected recipients. These and other findings suggested that the inability of circulating lymphocytes to gain access to lymphoid tissues in infected mice, coupled with the poor ability of those tissues to sequester parasite antigens, could account for the known prolonged delay in the development of curative antibody response characteristic ofT. musculi-infected mice. It is likely that the marked disruption of lymphoid tissue histoarchitecture that is typical ofT. musculiinfection contributes significantly to the failure of the tissues to sequester parasites and lymphocytes. Because lymphoid tissue disruption is seen in many parasitic infections, the findings reported here may have fairly broad relevance. In any case, the procedure described here for labeling parasites and lymphocytes should be of general utility for tracking their dispositionin vivo.

NOVEL SNAKE VENOM PROTEINS CYTOLYTIC TO CANCER CELLS IN VITRO AND IN VIVO SYSTEMS

Cancer cell inhibitors, named Atroporin and Kaotree, having molecular weights of 35 kDa and 6 kDa have been isolated from the venoms of Crotalus atrox and Naja naja kaouthia, respectively, by fractionation on high pressure liquid chromatography. The purified Atroporin and Kaotree showed killing effects on various types of human (breast, colon, liver, ovary, etc.) and animal cancer cells in concentrations as low as 0.5µg/ml, and having no effect on normal mouse kidney, liver, spleen, and erythrocytes up to 5.0µg/ml. Both Atroporin and Kaotree prevent the formation of ascitic tumors caused by myeloma cells in Balb/C mice. In addition, both Atroporin and Kaotree showed regression of ascitic tumors formed by myeloma cells. Atroporin and Kaotree complement each other, as in combination they showed elevated anti-cancer activity in vitro and in vivo systems. However, Atroporin and Kaotree are immunologically distinct proteins showing no cross reactivity. Atroporin and Kaotree, individually or in combination, have the potential for cancer biotherapy.

A population of CD62Llow Nk1.1- CD4+ T cells that resembles NK1.1+ CD4+ T cells.

In MHC class II-/- C57BL/6 (II-/-) mouse spleen, a small population of CD4+ T cells is present of which NK1.1+ CD4+ (NK) T cells comprise 40 to 45%. We report here that many of the NK1.1- CD4+ T cells derived from II-/- mice are also NK T cells. They produce large amounts of IL-4 in response to anti-CD3 ligation and do so without any requirement for the presence of IL-4 in the priming culture, a property characteristic of NK T cells. Their IFN-gamma production is large and is enhanced by IL-12. In addition, II-/- NK1.1- CD4+ T cells produce IL-4 as a result of culture with L cells expressing murine CD1 (L-CD1). We report that CD49b, a component of integrin VLA-2, is expressed on the majority of both NK1.1+ and NK1.1- NK T cells. NK1.1- NK T cells also exist in wild-type C57BL/6 mice. Evidence supporting this is that Vbeta8 usage by CD62Llow NK1.1- CD4+ T cells was approximately 5% higher than that by CD62Lhigh CD4+ T cells in wild-type mice in keeping with the estimated proportion of NK1.1- NK T cells in the CD62Llow population. CD62Llow CD4+ T cells from beta2-m(-/-) mice, which lack NK T cells, showed no increase in Vbeta8 usage. When activated by anti-CD3 or L-CD1, CD62Llow NK1.1- CD4+ T cells from conventional but not beta2-m(-/-) and CD1-/- mice produce IL-4 in a manner indistinguishable from II-/- NK1.1- CD4+ T cells. NK1.1- NK T cells in normal mouse spleens are approximately as numerous as NK1.1+ NK T cells.

Expression of Trypsin by Epithelial Cells of Various Tissues, Leukocytes, and Neurons in Human and Mouse

It has long been believed that trypsin is normally synthesized only in the pancreas. In the present study, expression of trypsin in human and mouse nonpancreatic tissues was examined. Northern blot analysis of normal human tissues indicated that the trypsin gene is expressed at high levels in the pancreas and spleen and considerably in the small intestine. However, in situ hybridization and immunohistochemistry demonstrated that trypsin is widely expressed in epithelial cells of the skin, esophagus, stomach, small intestine, lung, kidney, liver, and extrahepatic bile duct, as well as splenic and neuronal cells. In the spleen, trypsin message was detected in macrophages, monocytes, and lymphocytes in the white pulp. In the brain, it was detected in the nerve cells of the hippocampus and cerebral cortex. Analysis by gelatin zymography confirmed the presence of a latent or an active form of trypsin in various normal mouse tissues. Reverse transcription-polymerase chain reaction analysis also confirmed the expression of trypsin genes in the spleen, liver, kidney, and brain of normal mice. Such a broad distribution of trypsin suggests its general roles in the maintenance of normal epithelial cell functions, the immune defense system, and the central nervous system.

Cyclin D3 and megakaryocyte development: exploration of a transgenic phenotype.

The roles of cell cycle regulatory proteins in megakaryocyte development are poorly understood. We have previously demonstrated that cyclin D3 is expressed in megakaryocytes and is induced upon treatment with Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the megakaryocytic lineage show features similar to in vivo Mpl ligand treatment, including increased megakaryocyte number and ploidy. Terminal maturation and platelet production are not enhanced, however, and transgenic megakaryocytes show a defect in demarcation membrane development. We have examined expression of the transcription factor nuclear factor (NF)-E2, known to be involved in cytoplasmic maturation and platelet fragmentation, in these transgenic mice and controls treated with Mpl ligand. Our findings demonstrate marked induction of NF-E2 mRNA in control megakaryocytes in response to Mpl ligand, but no NF-E2 increase in transgenic cells, potentially explaining the lack of platelet increase in these transgenic mice. Transgenic megakaryocytes treated with Mpl ligand display a limited increase in NF-E2. In response to literature reports of Mpl ligand-induced transient increases in p21Cip1/WAF1 mRNA in polyploidizing megakaryocytic cell lines, we have examined p21 transcript levels in both normal and transgenic megakaryocytes. In normal mouse spleen, only a small percentage of megakaryocytes express detectable levels of p21 mRNA, with the majority of these cells expressing at high intensity. p21 levels are not affected by treatment with Mpl ligand, while the frequency of expressing cells increases transiently. Transgenic megakaryocytes exposed to Mpl ligand also show an increased frequency of p21-positive cells, and stimulation with Mpl ligand resulted in a further increase in this frequency. The nature of this effect will require further investigation.

Heterogeneity of Mouse Spleen Dendritic Cells: In Vivo Phagocytic Activity, Expression of Macrophage Markers, and Subpopulation Turnover

In the normal mouse spleen, two distinct populations of dendritic cells (DC) are present that differ in microanatomical location. The major population of marginal DC is found in the "marginal zone bridging channels" and extends into the red pulp. The interdigitating cells (IDC) are localized in the T cell areas in the white pulp. The aim of the present study was to characterize these two splenic DC populations with regard to their phenotype, in vivo phagocytic function, and turnover. Both marginal DC and IDC are CD11c+ and CD13+, but only IDC are NLDC-145+ and CD8alpha+. Notably, both populations, when freshly isolated, express the macrophage markers F4/80, BM8, and Mac-1. To study the phagocytic capacity of these cells, we employed the macrophage "suicide" technique by injecting liposomes loaded with clodronate i.v. Marginal DC, but not IDC, were eliminated by this treatment. Phagocytosis of DiI-labeled liposomes by DC confirmed this finding. The two DC populations differed significantly with regard to their turnover rates, as studied in a transgenic mouse model of conditional depletion of DC populations with high turnover. In these mice, marginal DC were completely eliminated, but the IDC population remained virtually intact. From these data we conclude that the marginal DC population has a high turnover, in contrast to the IDC population. Taken together, the present results indicate that marginal DC and IDC represent two essentially distinct populations of DC in the mouse spleen. They differ not only in location, but also in phenotype, phagocytic ability, and turnover.

Characterization of membrane-associated prothrombin activator in normal and injured murine tissues

Thrombin is a multifunctional enzyme involved in coagulation, cell modulation and inflammation. We recently reported a novel membrane-associated prothrombin activator, abbreviated as MAPA, found in cultured fibroblasts and glial cell lines. In this study, we examined the physiological role of this enzyme. MAPA-like activity was detected in the liver, kidney, lung and heart but not in the spleen or brain in normal mice. To examine whether MAPA participates in biological reactions, hepatic and renal injury were induced by administration of CCl 4 and HgCl 2, respectively. MAPA-like activity was specifically increased in the injured tissues: the activity was elevated by about 100-fold in 48 h in the liver and increased by about 5-fold in 12 h in the kidney. Their enzymatic properties were the same as those of MAPA in 8C feline kidney fibroblast cells. Phospholipids are required for activation of prothrombin by MAPA obtained from both 8C cells and tissues. These results suggest that MAPA activates prothrombin on the cell surface in injured tissue and participates in inflammation and regeneration associated with tissue injury.