Strain difference of murine bone marrow-derived mast cell functions

Mast cells play an important role for the induction and the expression of allergic responses. In this report, we studied the strain difference of bone marrow-derived murine mast cell (BMMC) functions in vitro. BMMC were induced by in vitro culture of bone marrow cells from BALB/c and C57BL/6 mice with interleukin (IL)-3 for 4 weeks, stimulated with immunoglobulin E antibody and antigen, and mediators and cytokines released in the culture supernatant were assayed. BMMC from C57BL/6 mice released a higher amount of granule-associated mediators, beta-hexosaminidase, and histamine than that from BALB/c mice. The expression of mRNA of histidine decarboxylase was higher in C57BL/6 mice. Conversely, the productions of newly synthesized mediators, prostaglandin D2 (PGD2), IL-6, and monocyte chemoattractant protein-1, and the mRNA expression of IL-5 were higher in BALB/c BMMC than C57BL/6 BMMC. Although mRNA and protein expression levels of cyclooxygenase-2 were equal in two strains, both expression levels of hematopoietic PGD synthase (hPGDS) were higher in BALB/c BMMC. Mast cells, freshly obtained from mice, also showed the same strain difference concerning the mediator release. These results indicate that the strain difference exists in mast cell functions in mice, and this difference can be considered to induce the susceptibility difference to allergic reactions in mouse strains.     

Altered in vitro apoptosis of cultured mast cells prepared from an inbred strain of mice, NC/Kuj

BACKGROUND: An inbred strain of mice, NC, develops dermatitis associated with highly elevated serum IgE and dermal mast cell hyperplasia. OBJECTIVES AND METHODS: To clarify the mechanisms for the dermal mast cell hyperplasia in NC, we prepared bone marrow-derived mast cells (BMMCs) from three strains of mice, NC/Kuj, C57BL/6 and BALB/c, and compare histamine contents, histamine release abilities, adhesive properties and apoptosis of the BMMCs. RESULTS: Compared with BMMCs obtained from C57BL/6 and BALB/c, NC/Kuj BMMC possessed higher histamine content and higher adhesive ability to plastic plates, although histamine release from BMMCs was found to be similar in the three strains. The most intriguing finding is the lack of apoptosis in the BMMCs from NC/Kuj upon growth factor deprivation as determined by DNA ladder formation and loss of membrane phospholipid asymmetry. CONCLUSION: The altered in vitro properties of mast cells in NC/Kuj partially account for an increase of dermal mast cells, which might be involved in the development of skin lesions in NC.     

Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient KitW-sh/KitW-sh sash mice

BACKGROUND: Mast cell-deficient Kit(W)/Kit(W-v) mice are an important resource for studying mast cell functions in vivo. However, because they are compound heterozygotes in a mixed genetic background and are infertile, they cannot be crossed easily with other mice. OBJECTIVE: To overcome this limitation, we explored the use of Kit(W-sh)/Kit(W-sh) mice for studying mast cell biology in vivo. RESULTS: These mice are in a C57BL/6 background, are fertile and can be bred directly with other genetically modified mice. Ten-week-old Kit(W-sh)/Kit(W-sh) are profoundly mast cell-deficient. No mast cells are detected in any major organ, including the lung. Gene microarrays detect differential expression of just seven of 16,463 genes in lungs of Kit(W-sh)/Kit(W-sh) mice compared with wild-type mice, indicating that resting mast cells regulate expression of a small set of genes in the normal lung. Injecting 10(7) bone marrow-derived mast cells (BMMC) into tail veins of Kit(W-sh)/Kit(W-sh) mice reconstitutes mast cell populations in lung, stomach, liver, inguinal lymph nodes, and spleen, but not in the tongue, trachea or skin. Injection of BMMC into ear dermis or peritoneum reconstitutes mast cells locally in these tissues. When splenectomized Kit(W-sh)/Kit(W-sh) mice are intravenously injected with BMMC, mast cells circulate longer and are found more often in the liver and inguinal lymph nodes, indicating that the spleen acts as a reservoir for mast cells following injection and limits migration to some tissues. CONCLUSION: In summary, these findings show that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice possess unique attributes that favour their use for studying mast cell functions in vivo.     

Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.     

Histamine-releasing properties of mast cells from various strains of mice

A comparison has been made of thein vitro histamine releasing capacity of peritoneal mast cells from BALB/c, C₃H/A, C57BL/6J and CFW mouse strains in response to immunological and non-immunological stimuli namely antimouse IgE, Concanavalin A (Con A), ionophore A23187 and sodium fluoride. Anti-IgE-induced histamine secretion was highest in mast cells of CFW mice, intermediate in the cells of C57BL/6J and C₃H/A mice and lowest in the cells of BALB/c mice. Similar, although less pronounced, strain-dependent differences were observed in Con A-induced release. Mast cells of C57BL/6J and CFW mice were significantly more sensitive to the action of ionophore A23187 compared to the two other strains, although the cells of each strain responded in different manner. Sodium fluoride-induced histamine release occurred in two ways: from mast cells of BALB/c and C57BL/6J it was dose-dependent at NaF concentrations of 1–15 mM, whereas cells of the two other strains were insensitive to the action of NaF at concentrations of 1–5 mM but at a concentration of 7.5 mM, a very strong potentiation of release was observed. Our results suggest functional heterogeneity of mast cells from various strains of mice, a point to be considered in further studies of mast cell function.This work was supported by the Polish Academy of Sciences (Grant No. 10.5).     

Mast cells at the host-pathogen interface: host-protection versus immune evasion in leishmaniasis

Infection of a susceptible host with Leishmania, a protozoan parasite, causes the disease leishmaniasis, which is characterized by neutrophil, eosinophil, macrophage, lymphocyte and mast cell infiltration into the infected tissue followed by parasite growth. Although the roles played by other cells in leishmaniasis are known, the role of mast cells remains to be ascertained. Here, we demonstrate that Leishmania regulates mast cell infiltration to the site of infection, mast cell production and mast cell function resulting in differential growth of the parasite in resistant (C57BL/6 or CBA/T6T6) and susceptible (BALB/c) macrophages. An interleukin-3-dependent augmentation in mast cell committed progenitors is observed in BALB/c but not in C57BL/6 mice during Leishmania infection. The mast cell supernatants inhibit IFN-gamma-dependent restriction of Leishmania growth in macrophages in BALB/c mice whereas the reverse phenomenon occurs in C57BL/6 mice. Our data reveals a different facet of host-pathogen interaction.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4⁺ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0×10⁷ cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-γ, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4⁺ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c×C57BL/6 F₁ mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-γ than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-γ-producing ability but rather derived from their poor responsiveness to IFN-γ.     

Specific and nonspecific desensitization of mouse mast cells

Peritoneal mast cells from BALB/c X C57BL/6I mice actively or passively sensitized with antiovalbumin and anti-hemocyanin IgE antibodies were desensitized in vitro by preincubation with antigen in suboptimal for histamine release conditions. Preincubation with an optimal antigen dose under calcium-free conditions induced rapid (15 min) complete desensitization of actively and passively sensitized cells to subsequent challenge with antigen under calcium-containing conditions. The responsiveness to anti-IgE under these conditions was unchanged in mast cells from passively and actively sensitized mice showing low level of IgE antibodies, whereas in the cells from mice showing very high IgE level partial desensitization to anti-IgE was observed (nonspecific desensitization). The results suggest the relationship between the level of mouse mast cell sensitization with IgE antibody and the mode of desensitization.     

Reversed anaphylaxis with anti-IgE on mouse and rat mast cells

Peritoneal mast cells of four inbred strains of mice (BABL/c, 129, C3H/A, C57BL/6J) and F1 hybrids of August and Wistar rats were studied for their sensitivity to the action of anti-IgE antibody for histamine release. The mast cells of normal mice from four mouse strains tested showed different abiltiy to release histamine after challenge with several dilutions of anti-IgE; mast cells of BALB/c mice showed the lowest reaction to anti-IgE. After active sensitization of mice and rats their mast cells release histamine after challenge with specific antigen, however, the detectable changes in their sensitivity to anti-IgE were not observed. After passive sensitization of mice the mast cells of three out of four strains tested show slightly enhanced sensitivity to anti-IgE for histamine release. Thus, the sensitization of mast cells not always stimulates their sensitivity to anti-IgE in reversed anaphylaxis.     

Mast cell-derived TNF contributes to airway hyperreactivity, inflammation, and TH2 cytokine production in an asthma model in mice

BACKGROUND: Mast cells, IgE, and TNF, which have been implicated in human atopic asthma, contribute significantly to the allergic airway inflammation induced by ovalbumin (OVA) challenge in mice sensitized with OVA without alum. However, it is not clear to what extent mast cells represent a significant source of TNF in this mouse model. OBJECTIVE: We investigated the importance of mast cell-derived TNF in a mast cell-dependent model of OVA-induced airway hyperreactivity (AHR) and allergic airway inflammation. METHODS: Features of this model of airway inflammation were analyzed in C57BL/6J-wild-type mice, mast cell-deficient C57BL/6J-Kit(W-sh)(/W-sh) mice, and C57BL/6J Kit(W-sh/W-sh) mice that had been systemically engrafted with bone marrow-derived cultured mast cells from C57BL/6J-wild-type or C57BL/6J-TNF(-/-) mice. RESULTS: Ovalbumin-induced AHR and airway inflammation were significantly reduced in mast cell-deficient Kit(W-sh/W-sh) mice versus wild-type mice. By contrast, Kit(W-sh/W-sh) mice that had been engrafted with wild-type but not with TNF(-/-) bone marrow-derived cultured mast cells exhibited responses very similar to those observed in wild-type mice. Mast cells and mast cell-derived TNF were not required for induction of OVA-specific memory T cells in the sensitization phase, but significantly enhanced lymphocyte recruitment and T(H)2 cytokine production in the challenge phase. CONCLUSION: Mast cell-derived TNF contributes significantly to the pathogenesis of mast cell-dependent and IgE-dependent, OVA-induced allergic inflammation and AHR in mice, perhaps in part by enhancing lymphocyte recruitment and T(H)2 cytokine production. CLINICAL IMPLICATIONS: Our findings in mice support the hypothesis that mast cell-derived TNF can promote allergic inflammation and AHR in asthma.     

Comparative study of American cutaneous leishmaniasis and diffuse cutaneous leishmaniasis in two strains of inbred mice.

Two Leishmania strains, AZV (isolated from a typical case of American cutaneous leishmaniasis) and AMP (from a case of diffuse cutaneous leishmaniasis), were studied in C57BL/6 and BALB/c mice. After infection with 10(4) amastigotes of either strain, C57BL/6 mice developed self-resolving lesions lasting 20 to 23 weeks and showed both delayed hypersensitivity response to leishmanial antigen and specific agglutinating antibodies. On the other hand, BALB/c mice infected with 10(4) AZV or AMP amastigotes developed chronic, large, ulcerated lesions and showed impaired cellular and humoral responses to the parasite. When BALB/c and C57BL/6 mice received 10(2) AMP amastigotes, patterns of infection were similar to those observed after inoculation of 10(4) amastigotes. In vitro studies revealed that spleen cells from AZV- or AMP-infected C57BL/6 mice showed an increased DNA-synthetic response to leishmanial antigen, concanavalin A, and phytohemagglutinin. Spleen cells from AZV- or AMP-infected BALB/c mice showed an increased response to concanavalin A and diminished responses to leishmanial antigen, phytohemagglutinin, and lipopolysaccharide.     

Is the follicular dendritic cell a primarily stationary cell?

The cell nature of follicular dendritic cells (FDC), a member of dendritic cell group, was examined to see whether or not they are recirculating cells on splenic implantation study. Slices of BALB/c mouse spleen were implanted into C57BL/6 mice neonatally thymectomized and reconstructed by F1 (BALB/c x C57BL/6) thymus grafting. On H-2 class I immunohistology 6 months later, host spleens consisted of only the cells including FDC of host (C57BL/6) type. On the other hand, FDC of regenerated splenic grafts were of splenic donor (BALB/c) origin and haematogenic cells including germinal centre lymphocytes were of the host (C57BL/6) origin. The fact that the FDC in the regenerated splenic grafts reside irrespective of the replacement by host recirculating cells indicates that FDC belong primarily to stationary cell populations but not recirculating cell populations.     

Beige mouse mast cells generated in vitro: ultrastructural analysis of maturation induced by sodium butyrate and of IgE-mediated, antigen-dependent degranulation

We derived growth factor-dependent mast cell lines from C57BL/6-bgJ/bgJ ('beige') mouse bone marrow cells using techniques previously described for deriving mast cell lines from normal mice. According to examination by transmission electron microscopy, the cytoplasmic granules of cultured mast cells derived from beige mice were larger in size and fewer in number than those in cultured mast cells derived from normal C57BL/6 mice. Mast cells derived from beige mice underwent maturation when exposed to sodium butyrate, as judged by increased content of electron-dense material in the cytoplasmic granules. Cultured mast cells derived from beige mice also underwent IgE-mediated, antigen-dependent anaphylactic degranulation which was similar in its ultrastructural features to that described for cultured mast cells from normal mice. However, in mast cells not stimulated with IgE and antigen, fusion between individual cytoplasmic granules was observed more commonly in mast cells derived from beige mice than in normal mast cells. This might mean that these events are more common in C57BL/6-bgJ/bgJ mast cells, and/or that their resolution is slower and thus more easily captured by electron microscopy.     

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.     

Amastigote load and cell surface phenotype of infected cells from lesions and lymph nodes of susceptible and resistant mice infected with Leishmania major

Cells of the dendritic cell (DC) lineage, by their unique ability to stimulate naive T cells, may be of crucial importance in the development of protective immune responses to Leishmania parasites. The aim of this study was to compare the impact of L. major infection on DCs in BALB/c (susceptible, developing Th2 responses), C57BL/6 (resistant, developing Th1 responses), and tumor necrosis factor (TNF)(-/-) C57BL/6 mice (susceptible, developing delayed and reduced Th1 responses). We analyzed by immunohistochemistry the phenotype of infected cells in vivo. Granulocytes (GR1(+)) and macrophages (CD11b(+)) appear as the mainly infected cells in primary lesions. In contrast, cells expressing CD11c, a DC specific marker, are the most frequently infected cells in draining lymph nodes of all mice tested. These infected CD11c(+) cells harbored a particular morphology and cell surface phenotype in infected C57BL/6 and BALB/c mice. CD11c(+) infected cells from C57BL/6 and TNF(-/-) C57BL/6 mice displayed a weak parasitic load and a dendritic morphology and frequently expressed CD11b or F4/80 myeloid differentiation markers. In contrast, some CD11c(+) infected cells from BALB/c mice were multinucleated giant cells. Giant cells presented a dramatic accumulation of parasites and differentiation markers were not detectable at their surface. In all mice, lymph node CD11c(+) infected cells expressed a low major histocompatibility complex II level and no detectable CD86 expression. Our results suggest that infected CD11c(+) DC-like cells might constitute a reservoir of parasites in lymph nodes.     

Morphofunctional characteristic of mast cells in BALB/c and C57Bl/6 mice during cold exposure

Mast cells of the mesentery and subcutaneous tissue in BALB/c and C57Bl/6 mice were studied after single and repeated cold exposure (−20°C, 3 min). Immediate adaptive reactions of mast cells in BALB/c and C57Bl/6 mice did not differ after single cold exposure and were manifested in increased degranulation. Repeated cold exposure of BALB/c mice was followed by an adaptive reaction, which included an increase in the count of mast cells in subcutaneous tissue and normalization of the degranulation index. In C57Bl/6 mice the count of mast cells in subcutaneous tissue decreased, while the degranulation index remained high. These changes reflect the disadaptive response of mast cells to repeated cold exposure. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 8, pp. 207–209, August, 2004     

Correlation between natural killer cell activation in the bone marrow and haemopoietic dysfunction following cytomegalovirus infection of mice.

We describe here the activation of natural killer (NK) cells in the bone marrows and spleens of mice infected with murine cytomegalovirus (MCMV). NK activity at these sites peaked at day 2 to 3 post-infection (p.i.) and declined between days 6 and 10 p.i. in BALB/c and C57BL/6 mice. In BALB/c mice, the increases in NK activity coincided with depletion of colony-forming units of the granulocyte-monocyte lineage (CFU-GM) from the marrow. CFU-GM depletion in MCMV-infected C57BL/6 mice was less severe, despite the presence of activated NK cells in the marrow. Treatment of BALB/c mice with anti-asialo GM1 prior to MCMV infection resulted in less severe CFU-GM depletion at day 2 p.i. than infection with MCMV alone. When homozygous C57BL/6 or CBA/CaH bg/bg mice were infected with MCMV, depletion of marrow CFU-GM was more severe than in their heterozygous littermates. Finally, we observed some inhibition of colony formation when marrow cells from MCMV-infected and uninfected BALB/c donors were mixed and incubated prior to the CFU-GM assay. These results suggest that activated NK cells may contribute to depletion of haemopoietic cells soon after MCMV infection of BALB/c mice, but may limit the loss of these cells in C57BL/6 and CBA/CaH mice.     

Dendritic cell populations in Leishmania major-infected skin and draining lymph nodes

Using a metacyclic promastigote ear infection model of cutaneous leishmaniasis, we examined the phenotype, parasite load, and cytokine production of dendritic cells in the skin and draining lymph nodes of resistant C57BL/6J and susceptible BALB/c mice. Five dendritic cell populations were isolated from the skin and lymph nodes, and the main difference between the groups of mice was an increased number of plasmacytoid dendritic cells in the lymph nodes of the susceptible mice. Although similar cell types were present in the skin emigrants of both strains, there was a 10-fold larger number of cells in BALB/c mouse skin early in infection than in C57BL/6J mouse skin. None of the dendritic cells in the lymph nodes harbored parasites until 3 weeks after infection, with the Langerhans cells having the largest load and the plasmacytoid dendritic cells having the smallest load but the longest lasting infection. Although parasites could be detected in the lymph nodes a few hours after infection, none of the skin emigrants harbored parasites, indicating that they are not the vehicle that ferries the parasites from the skin to the lymph nodes. The presence of larger numbers of plasmacytoid cells in infected BALB/c mice, the more protracted infection of these cells, and their production of alpha interferon point to a complex and important role for the plasmacytoid cells in leishmaniasis.