Concanavalin A-stimulated Spleen Cells Research Articles

Investigation on prescription screening of a polyherbal preparation based on splenocyte proliferation activity and its preparation method

Objective: The present study aims to optimize the extraction conditions of polysaccharides and flavonoids from a polyherbal preparation consisting of three kinds of Chinese medicinal herbs, Codonopsis pilosula, Crataegus pinnatifida, and Lycium barbarum, and evaluation of its immunomodulatory activity in immunosuppressed mice. Materials and Methods: An orthogonal design (L9 [3]4) was constructed to achieve the optimal extraction conditions. The immunomodulatory action of the polyherbal preparation was studied at three doses of 10, 20, and 40 mL/kg/day orally by measuring splenocyte proliferation in mice model of cyclophosphamide-induced immunosuppression. Results: The chosen parameters, including the ratio of solvent-to-raw material, duration of extraction, and extraction times, were the fundamental variables that influenced the extract yields. The highest yield of total polysaccharides content was 54.3 mg/mL when the ratio of solvent-to-raw material, duration of extraction, and number of extractions were 12:1, 1.5 h, and 3, respectively. The maximum extraction yield of the flavonoids was 3.5 mg/mL when the ratio of solvent-to-raw material was 12:1, the extraction time was 2 h, and the number of extractions was 3. The prescription screening showed that the impact of the polyherbal preparation on the splenocyte proliferation capacity was more pronounced than its disassembled components. Oral administration of the polyherbal preparation could significantly increase the concanavalin A-stimulated mouse spleen cells proliferation in a dose-dependent manner. Conclusion: These findings suggested that the polyherbal preparation possesses potential for augmenting the immune activity due to the polysaccharide and flavonoid content in these herbal medicines.

Anti-inflammatory effect of resveratrol on adjuvant arthritis rats with abnormal immunological function via the reduction of cyclooxygenase-2 and prostaglandin E2

Rheumatoid arthritis (RA) is a chronic inflammatory disease with unknown etiology. The present study investigated the anti-inflammatory effect of resveratrol on rats with adjuvant arthritis (AA) with abnormal immunological function via the reduction of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). AA model rats were established by injection of complete Freund's adjuvant and alterations in the rats secondary paw swelling and the polyarthritic scores were observed. Pathological examination of joint tissues was observed by hematoxylin and eosin staining. The proliferation of spleen cells was examined using a 3-(4,5-dimethylthiazol-2‑yl)-2,5-diphenyltetrazolium bromide assay in vitro. The protein expression of COX-2 in the synovial tissues was detected by western blotting. The level of PGE2 in the serum was assayed using an ELISA kit. The results demonstrated that resveratrol (10 or 50 mg/kg) was able to significantly reduce paw swelling and decrease the arthritis scores. Compared with the AA model rats, a significant reduction in the proliferation of concanavalin A-stimulated spleen cells was observed, articular cartilage degeneration with synovial hyperplasia and inflammatory cell infiltration was suppressed and the production of COX-2 and PGE2 in AA rats was reduced by treatment with resveratrol. These results suggest that resveratrol has significant anti-inflammatory effects on AA rats, which may be associated with the reduction of COX-2 and PGE2 inflammatory mediators.

Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats

Because of its antibacterial activity nanosilver is one of the most commonly used nanomaterials. It is increasingly used in a variety of both medical and consumer products resulting in an increase in human exposure. However, the knowledge on the systemic toxicity of nanosilver is relatively limited. To determine the potential systemic toxicity of silver nanoparticles (Ag-NP) with different sizes (20 nm and 100 nm) a 28-days repeated dose toxicity study was performed in rats using intravenous administration. The toxic effect of the 20 nm Ag-NP was performed using the bench mark dose (BMD) approach.Treatment with a maximum dose of 6 mg/kg body weight was well tolerated by the animals. However, both for 20 nm and 100 nm Ag-NP growth retardation was observed during the treatment. A severe increase in spleen size and weight was present which was due to an increased cell number. Both T and B cell populations showed an increase in absolute cell number, whereas the relative cell numbers remained constant. At histopathological evaluation brown and black pigment indicating Ag-NP accumulation was noted in spleen, liver, and lymph nodes. Ag-NP was also detected incidentally in other organs. Clinical chemistry indicated liver damage (increased alkaline phosphatase, alanine transaminase, and aspartate transaminase) that could not be confirmed by histopathology. Hematology showed a decrease in several red blood cell parameters.The most striking toxic effect was the almost complete suppression of the natural killer (NK) cell activity in the spleen at high doses. Other immune parameters affected were: decreased interferon-γ and interleukin (IL)-10 production by concanavalin-A stimulated spleen cells, increased IL-1β and decreased IL-6, IL-10 and TNF-α production by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. For the spleen weight a critical effect dose of 0.37 mg/kg body weight (b.w.) could be established. The lowest critical effect dose (CED) for a 5% change compared to control animals was observed for thymus weight (CED05 0.01 mg/kg b.w.) and for functional immune parameters, i.e. decrease in NK cell activity (CED05 0.06 mg/kg b.w.) and LPS stimulation of spleen cells (CED05 0.04 mg/kg b.w.). These results show that for nanosilver the most sensitive parameters for potential adverse responses were effects on the immune system.

H1521, a novel derivative of 4-hydroxyquinoline-3-carboxamide, suppresses the development of lupus in mice by inducing Th1 cytokine profile in T cells

Transferring parental splenocytes into unirradiated F1 mice induces a chronic graft-versus-host disease (GVHD), characterized by the production of Th2 cytokines and immunocomplex-mediated glomerulonephritis resembling systemic lupus erythematosus (SLE). The effects of H1521, a new derivative of 4-hydroxyquinoline-3-carboxamide, were investigated in chronic GVHD lupus model. H1521 was administered to chronic GVHD mice for 10 weeks. Nephritic symptoms were monitored and cytokine expression in the spleen was detected. To clarify the direct effect of H1521 on CD4 + T cell, CD4 + T cells were isolated and co-cultured with H1521 under neutral and Th1 or Th2 driving conditions in vitro. H1521 (32 mg/kg) reduced the incidence of proteinuria by 50% in chronic GVHD mice. Ameliorated lupus symptoms and improved renal histopathology damage were also observed. Administration of H1521 had little impact on Th1 cytokine IL-2 and IFN-gamma or Th2 cytokine IL-4 and IL-10 mRNA expression. In contrast, severely deficient IFN-gamma production by concanavalin A-stimulated spleen cells in chronic GVHD mice was completely restored by H1521. In accordance with this, decreased T-bet mRNA expression became normalized with H1521 (32 mg/kg) treatment. In addition, in vitro studies demonstrated that H1521 preferentially favored Th1 differentiation in CD4 + T cell and promoted IFN-gamma secretion in Th1 differential CD4 + T cell. However, IL-4 secretion in naive or Th2 differential CD4 + T cell was unaffected by H1521. In conclusion, H1521 can induce Th1 cytokine profile in CD4 + T cells and has possible therapeutic value in Th2-predominant immune diseases.

Innate profiles of cytokines implicated on oral tolerance correlate with low‐ or high‐suppression of humoral response

Oral tolerance (OT) is being studied with great interest because of its therapeutic potential in allergy and autoimmunity. In the present study, two mouse strains with extreme phenotypes of OT susceptibility (TS) or resistance (TR) to ovalbumin (OVA) were used to demonstrate whether the tr and ts genes, cumulated during 18 generations of bi-directional genetic selection, influence expression of immunobiological traits in naive or antigen-gavaged TR/TS mice. The difference in anti-OVA titres was 2048-fold between OVA-gavaged TS and TR mice. Tolerance susceptibility to OVA gavage in individuals from a (TS x TR)F(2) population was 24% high-susceptibility, 62% low-susceptibility and 14% non-tolerant. Different antigens, unrelated to OVA, were tested by gavage and TS mice were generally susceptible while TR mice were resistant. The stability of TS and TR phenotypes was not affected by the use of strict protocols of intraperitoneal immunization or feeding over 30 consecutive days. The levels of interleukin-2 (IL-2), IL-4, interferon-gamma and IL-10 cytokines evaluated in concanavalin A-stimulated spleen cells from naive mice and in OVA-stimulated spleen cells from OVA-gavaged mice were higher in TS mice. Interleukin-10 was up-regulated in OVA-gavaged TS mice and down-regulated in TR mice. In naive mice, the percentage of CD4(+) CD25(+) and CD4(+) Foxp3(+) spleen cells and IL-10 expression by CD4(+) cells was significantly higher in TS mice. These results indicate that regulation of IL-10 expression could be an important factor contributing to the mechanisms controlling OT susceptibility, and that the OT responses of TR and TS individuals strongly correlate with their innate potential to secrete this cytokine.

Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.

Induction and expression of protective T cells during Mycobacterium avium infections in mice.

Mycobacterium avium is an opportunistic pathogen that infects individuals suffering from chronic lung disease or immunocompromised patients such as AIDS patients. Here we show that a highly virulent isolate of M. avium proliferated as extensively in T cell deficient as in immunocompetent mice. T cell deficient mice allowed a progressive growth of a less virulent AIDS-derived isolate of M. avium while immunocompetent mice arrested the growth of this isolate. Adoptive transfer of T cell enriched spleen cells between congenic strains of mice differing at the Bcg/Ity/Lsh locus showed that only naturally resistant BALB/c.Bcgr (C.D2) mice infected with the highly virulent strain of M. avium or the naturally susceptible BALB/c mice infected with the lower virulence isolate developed protective T cells and that these cells only mediated protection when transferred to naturally susceptible, but not to naturally resistant, mice. Both strains of M. avium proliferated in bone marrow-derived macrophages cultured in vitro and they were both susceptible to the bacteriostatic effects induced in the macrophages by crude lymphokines produced by concanavalin A-stimulated spleen cells.

Effects of boar seminal immunosuppressive fraction on production of cytokines by Concanavalin A-stimulated spleen cells and on proliferation of B lymphoma cell lines.

The immunosuppressive fraction (ISF) of boar seminal vesicle fluid has recently been demonstrated to inhibit mitogen-stimulated proliferation of lymphocytes and antibody response to corpuscular and soluble antigens. The effects of ISF on in vitro and in vivo production of cytokines as well as its possible inhibitory effect on proliferation of B lymphoma cells remain to be elucidated. The effect of ISF on proliferation of normal mouse spleen cells stimulated by Concanavalin A (Con A) and on mouse B lymphoma cells was measured by 3H-thymidine incorporation. Cytokines were determined in the supernatants of mouse spleen cells stimulated with Con A in the presence or absence of ISF by enzyme-linked immunosorbent assay (ELISA). In vivo cytokine production in the sera samples of mice treated with ISF and immunized with keyhole limpet hemocyanin (KLH) was followed by ELISA, too. We confirmed the inhibitory effect of ISF on Con A-stimulated lymphocyte proliferation. ISF affected cytokine production in the Con A-stimulated spleen cells: production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was lowered, but production of IL-4, IL-6, and IL-10 was enhanced. Similarly, in the sera samples of mice immunized with keyhole limpet hemocyanin (KLH), IL-2 and IFN-gamma levels were decreased by ISF. ISF inhibited proliferation of Ag 8 and X 63-IL-2 B lymphoma cells as well. ISF inhibited production of T helper1 (Th1) cytokines (IL-2 and IFN-gamma) and enhanced production of Th2 cytokines (IL-4, IL-6, and IL-10). ISF seems to shift the Th1/Th2 pattern in favor of Th2. ISF exhibited an antiproliferative activity on mouse B lymphoma cells.

Salivary gland extract from Ixodes ricinus tick modulates the host immune response towards the Th2 cytokine profile.

In our previous work, the salivary gland extract (SGE) from Ixodes ricinus ticks impaired T-lymphocyte proliferation and clearly modulated the immune response towards the Th2 pattern in human peripheral blood mononuclear cell culture. In the present work, the results obtained on mouse splenocytes are compared with those on human leukocytes. ELISA (protein level) and RNAse protection assay (mRNA level) showed that SGE enhanced interleukin (IL)-1alpha, IL-1beta, IL-1Ra, IL-6, and IL-12p40 cytokines, whereas production of IL-2, IL-5, IL-10, and IL-13 was decreased. The minute levels of IL-9, IL-15 and IL-12p70 were not changed after the addition of tick saliva. IL-4 was upregulated, whereas the production of gamma interferon and migratory inhibition factor was downregulated after the addition of SGE. Tick saliva decreased concanavalin A-stimulated spleen cell proliferation and the percentage of activated T-cells. We conclude that the Th2 polarization did not involve all of the cytokines tested. However, the Th2 subset-augmenting effect of tick saliva was confirmed.

Cloning, expression and purification of recombinant cotton rat interferon-gamma.

The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.

CYTOKINE PRODUCTION BY C57BL/6 MOUSE SPLEEN CELLS IS SELECTIVELY REDUCED BY EXPOSURE TO PROPANIL

Numerous immunomodulatory effects are caused by propanil, an extensively used postemergent herbicide. The T-dependent antibody response is suppressed after exposure to propanil, raising the question of propanil's effect on T-helper-cell populations. In the present study, we show that the production of several T-cell cytokines is affected by propanil after in vivo or in vitro exposure. In vivo exposure to propanil caused the reduction of interleukin (IL)-2, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)- production in concanavalin A-stimulated spleen cell cultures established 2 d after exposure. IFN- and GM-CSF production had recovered by d 4 postexposure; however, IL-2 and IL-6 levels continued to be depressed through d7 postexposure. Continuous in vitro treatment of normal spleen cells with propanil decreased IL-2, IL-6, GM-CSF, and IFN- production after concanavalin A activation. Pulsing normal spleen cell cultures with propanil for up to 8 h before T-cell activation resulted in reduced IL-6 but not IL-2 or IFN- production. These data indicate that propanil can selectively inhibit spleen cell cytokine production, which could contribute to the immunomodulatory effects previously described.

Inhibition of Eimeria tenella Development in vitro Mediated by Chicken Macrophages and Fibroblasts Treated with Chicken Cell Supernatants with IFN-g Activity

Pretreatment of chicken macrophages or fibroblasts with supernatants from concanavalin A-stimulated spleen cells or from the virus-transformed cell line reticuloendotheliovirus as source of interferon gamma (IFN-gamma) slows down subsequent sporozoite replication in the cells. To identify the presence of IFN-gamma, we combined four typical activities of IFN-gamma: inhibition of cytopathic effect of vesicular stomatitis virus on IFN-gamma-treated fibroblasts, cytostatic activity of IFN-gamma-activated macrophages, induction of major histocompatibility complex II antigen expression on IFN-gamma-activated fibroblasts and macrophages, and induction of nitrite production in macrophages. We have shown that chicken fibroblasts and macrophages possess a microbiostatic capacity once they are able to prevent the otherwise unchecked intracellular replication of Eimeria tenella following activation with culture supernatants identified as containing a strong IFN-gamma activity.

Apoptosis and in vivo distribution and clearance of eosinophils in normal and Trichinella spiralis-infected rats.

We examined apoptosis of eosinophils obtained from normal rats and rats infected with Trichinella spiralis (T. spiralis). A considerable percentage of normal rats underwent apoptosis within a few hours, however, the apoptosis rate of infected rats was significantly lower. This rate began decreasing only 12 h after infection, reached its lowest level on day 3, and was restored to normal by day 40. We also studied the effects on eosinophil apoptosis of culture supernatants from concanavalin A-stimulated spleen cells from infected rats and found that supernatants from days 3, 7, and 18 of infection inhibited apoptosis eosinophils from normal rats. We then examined the distribution and clearance of eosinophils in vivo. In normal rats, the largest numbers of eosinophils were found in the spleen, liver, and bone marrow. In the infected rats, however, by day 3, by which time the worms had begun to migrate, there was a striking increase in these cells in the intestines. In summary, normal eosinophils are unexpectedly sensitive to apoptosis, whereas infected eosinophils become resistant to this form of cell death.

Chronic Pseudomonas aeruginosa lung infection is more severe in Th2 responding BALB/c mice compared to Th1 responding C3H/HeN mice

The chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) is characterized by a pronounced antibody response and microcolonies surrounded by numerous polymorphonuclear neutrophils (PMN). Poor prognosis is correlated with a high antibody response to P. aeruginosa antigens. An animal model of this infection was established in two strains of mice: C3H/HeN and BALB/c, generally known as Th1 and Th2 responders, respectively, which were challenged with alginate-embedded P. aeruginosa. Mortality was significantly lower in C3H/HeN compared to BALB/c mice (p < 0.025). P. aeruginosa was cleared more efficiently in C3H/HeN mice and significantly more C3H/HeN mice showed normal lung histopathology (p < 0.02), and we found significantly fewer microabscesses in C3H/HeN mice than in BALB/c mice (p < 0.005). In supernatants from P. aeruginosa antigen and concanavalin A-stimulated spleen cells from the two strains of mice, the interferon-(IFN-) gamma levels were higher, whereas IL-4 levels were lower in C3H/HeN mice than in BALB/c mice. The implications of these findings for CF patients with chronic P. aeruginosa lung infection are discussed.

Parasite Antigen-Induced IFNg and IL-4 Production by Cells from Pathopermissive and Pathoresistant Strains of Mice Infected with Trypanosoma cruzi

The cardiac pathology associated with infection by Trypanosoma cruzi in mice has been suggested to be partially dependent upon cytokine responses. The pathoresistant B10.D2 mice, which display little infection-induced myocarditis, and the pathopermissive DBA/2 mice, which show significant cardiac damage, were compared for their in vitro interferon (IFN)-gamma and interleukin (IL)-4 production. Concanavalin A-stimulated spleen cells from infected B10.D2 mice produce a greater amount of IFN-gamma than DBA/2 mice, whereas the IL-4 production is only slightly greater in the B10.D2 mice than the DBA/2 mice. Parasite antigen stimulation of spleen cells from these mice results in a clearly greater IFN gamma production by the B10.D2 and a higher IL-4 level for the DBA/2 mice. The data presented suggest a relationship between an enhanced TH1-type response and decreased chronic cardiac pathogenesis, whereas a lower level of TH1 activity may play a role in cardiac involvement.

An Antiviral Effect of Nitric Oxide: Inhibition of Reovirus Replication

We have previously shown that macrophages from chickens infected with avian reovirus are primed to produce nitric oxide (NO) in response to T cell cytokines and bacterial lipopolysaccharide (LPS). We now show that NO exerts potent antireovirus effects. Reovirus replication was substantially reduced in a chicken macrophage cell line, HD11, induced to make NO by stimulation with LPS or conditioned medium from concanavalin A-stimulated spleen cells. The use of a competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine, reduced the antiviral effect of LPS-stimulated HD11 cells. Cytostatic effects were concurrent with the observed antiviral effects of NO. Among these cytostatic effects were reduction in DNA synthesis, protein synthesis, and mitochondrial metabolism. These results indicated that a potential consequence of macrophage priming following virus infection is the protection of cells against virus-induced replication and cytopathic effects, and this protection may be mediated by the cytostatic effects of NO on the host cell.

Bop: a new T-cell-restricted gene located upstream of and opposite to mouse CD8b.

In the course of transient expression studies undertaken to determine the location of the mouse CD8b gene promoter, two additional promoter activities were detected within 600 nucleotides upstream of the gene. One activity directs transcription in the same direction as CD8b but fails to transcribe the CAT reporter gene due to an apparent transcription-blocking element lying between it and the gene. The second activity directs transcription opposite to that of the CD8b gene. Northern hybridization with a probe consisting of nucleotides -875 to -550 relative to the site of CD8b transcription initiation revealed hybridizing species of 4 kilobases (kb) and 1.8 kb in poly-A-selected RNA from mouse thymus but not from any other tissues. Similar RNA species were detected in poly-A+ RNA from concanavalin A-stimulated spleen cells and several long-term CTL lines but not from the EL4 or BW5147 T-cell lines or the J558L myeloma. The mRNA species were most abundant in cells of a secondary mixed leukocyte culture which were greater than 95% CD8(+). Northern hybridizations using single-stranded unidirectional probes indicated that these mRNAs represent transcription opposite to the CD8b gene. The tissue and cell type distribution of this newly-discovered gene (designated Bop for CD8b opposite) are consistent with T-cell-specific and possibly CD8-positive T-cell-specific expression. The head-to-head arrangement of the Bop and CD8b genes is reminiscent of the arrangement of the Tap1 and Lmp2 genes, and the expression of the Bop gene in CD8-positive cells raises the possibility that these genes are involved in the same functional pathway.

Infection With Nippostrongylus brasiliensis Induces Invasion of Mast Cell Precursors From Peripheral Blood to Small Intestine

Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtained from the bone marrow, peripheral blood, and small intestine of Ws/Ws rats with a small deletion at the tyrosine kinase domain of c-kit and of control normal (+/+) rats. In the culture containing concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM) alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC. In the culture containing both ConA-SCM and stem cell factor (a ligand of c-kit), however, the numbers of mast-cell colonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC. Using this culture condition, we investigated changes in concentration of CFU-mast in the marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in S phase of the cell cycle remained at low levels in the marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site.

Characterization of Cultured Mast Cells Derived From Ws/Ws Mast Cell-Deficient Rats With a Small Deletion at Tyrosine Kinase Domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP-I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I-/II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA-SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

Characterization of cultured mast cells derived from Ws/Ws mast cell- deficient rats with a small deletion at tyrosine kinase domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.