Appearance of mast cells in bone marrow, peripheral blood and spleen of immunized rats

The appearance of mast cells and their progenitors was studied in rats. The animals were immunized according to a protocol utilizing 2-week periods of daily administration of small doses of ovalbumin either as aerosol or as subcutaneous injection without adjuvant. These procedures are known to induce allergic responses of the IgE-mediated type. The immunization procedures resulted in increased numbers of mature mast cells in bone marrow. There were also increased numbers of progenitors, as indicated by increased maturation of mast cells in cultures of bone marrow cells from immunized animals compared with nonimmunized controls. Bone marrow cells in cultures from immunized and nonimmunized animals showed similarly increased maturation of mast cell progenitors when stimulated with conditioned medium from mixed lymphocyte reaction cultures. The number of progenitors was considerably lower in peripheral blood and spleen than in bone marrow. However, cultures of cells from peripheral blood and spleen of aerosol-immunized rats contained cells which matured into mast cells after addition of medium from mixed lymphocyte reaction cultures. The study shows that in immune responses similar to those seen in allergic individuals mast cells are normal constituents.     

In vitro studies on mast cell proliferation in N. brasiliensis infection.

We have previously shown that mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) proliferate and mature in rat bone marrow cultures stimulated with factors from antigen or mitogen-activated T lymphocytes. Here we have used this system to explore the MMC hyperplasia which occurs in infections with gastrointestinal nematode parasites. Lymphocytes producing MMC-growth factor were present from day 10 onwards in N. brasiliensis-infected rats and mesenteric lymph nodes (MLN) were the major source of activated lymphocytes. When different tissues of normal rats were cultured in the presence of conditioned medium by far the greatest proliferation of MMC occurred in bone marrow, indicating an origin of MMC from haemopoietic precursors. Cultures of infected rat bone marrow yielded considerably greater numbers of MMC than cultures of normal rat bone marrow and experiments using semisolid culture media indicated that N. brasiliensis infection causes an increase in the frequency of MMC progenitors in the bone marrow. A scheme is put forward for the sequence of events occurring in vivo based on the results of these and other published experiments. The reasons for the restricted in vivo localization of MMC to the mucous membranes and associated lymph nodes is discussed. Finally we give the results of microspectrophotometric analysis which has shown that the cultured mast cell contain a non-heparin proteoglycan, thus adding a further feature to the list of MMC-like properties of these cells.     

Adoptive transfer of resistance to Nematospiroides dubius infections of mice and an assay to measure the in vitro proliferation of lymphocytes reactive with N. dubius antigen.

Spleen and mesenteric lymph node cells from Nematospiroides dubius-infected and normal control mice were cultured in vitro with N. dubius antigen. Proliferation of these cells in response to antigen was measured by the uptake of [3H]TdR. Cells harvested from mice during a primary infection did not proliferate in vitro; however, low levels of specific proliferation could be demonstrated if these mice were treated on Day 5 post-infection with 20 mg/kg of cyclophosphamide i.p. A strong cell proliferative response was measured 6 days following a challenge infection; spleen cells responded more strongly than cells from the mesenteric lymph nodes (MLN), but the addition of lymph node cells to spleen cell cultures did not suppress the latter response. Responsiveness of spleen cells to concanavalin A (Con A) was two-fold higher in infected mice than in normal controls, but the proliferation of MLN cells to Con A was similar in infected and uninfected mice. When N. dubius-resistant B10.M (H-2f) mice were compared to the susceptible B10.BR (H-2k) mice, no differences were observed in the spleen cell response to N. dubius adult antigen following challenge infections. However, after a tertiary infection, MLN cells from the resistant strain proliferated strongly in comparison to cells from susceptible mice. Spleen or MLN cells from resistant mice transferred immunity to naive recipients provided that the recipients had received a prior injection i.p. with adult N. dubius antigen. The injection alone, or cells in the absence of the injection, failed to protect the recipients from N. dubius challenge.     

Bone marrow origin of mucosal mast cells

Infection with the intestinal parasite Nippostrongylus brasiliensis stimulates an accumulation of mucosal mast cells (MMC) in the villi of the small intestine of normal but not athymic or W/Wv anemic mice. W/Wv mice are congenitally deficient in both MMC and skin and connective tissue mast cells (CTMC). Athymic mice have normal or elevated numbers of CTMC but are severely deficient in MMC. CTMC derive from the bone marrow. To determine the origin of MMC, athymic and W/Wv mice were given various hematopoietic or lymphoid tissues from normal littermate or beige mice and the MMC response to N. brasiliensis infection was evaluated. The MMC defect in athymic mice was repaired by grafts of thymus cells, thymus gland, or spleen cells, but not by bone marrow cells or anti-Thy 1-treated bone marrow or spleen cells. The MMC and CTMC defects of W/Wv mice were repaired by grafts of bone marrow, spleen cells, or anti-Thy 1-treated bone marrow or spleen cells. Neither the MMC nor the CTMC defect in W/Wv mice was repaired by grafts of thymus cells or thymus glands. These results indicate the following, MMC, like CTMC, derive from the bone marrow and not from the thymus. MMC require a thymic influence for development. Athymic mice possess bone marrow precursors for both MMC and CTMC but lack a thymus-dependent component necessary for MMC development. W/Wv mice lack both MMC and CTMC mast cell precursors but possess the thymus-dependent component required for MMC development.     

Mast cell differentiation in cultures of T cell-depleted mesenteric lymph node cells from Nippostrongylus brasiliensis-infected mice

Lymphoid and bone marrow cells from normal and horse serum-immunized mice and lymphoid cells from Nippostrongylus brasiliensis-infected mice were cultured on monolayers of embryonic skin fibroblasts to analyze the factors which regulate the differentiation and proliferation of mast cells in vitro. Our results indicate that T cells can regulate the development of mast cells in vitro by either enhancement or suppression. In cultures of horse serum-immune spleen cells, inducer T cells are required for mast cells to develop. However, in cultures of mesenteric lymph node cells from N. brasiliensis-infected mice, inducer T cells are not required for mast cell development. This suggests that the development of mast cells may occur at discrete interleukin-3 (IL-3)-dependent and IL-3-independent stages. Mast cell precursors in the mesenteric lymph nodes of N. brasiliensis-infected mice may have already been acted on by inducer cells in vivo to become mast cell committed. While IL-3 does not appear to be required for mast cell development, these precursors do require the presence of a connective tissue microenvironment such as embryonic skin. The precursors can be inhibited from development by interferon preparations.     

Sequential appearance of basophils and mast cells from human bone marrow in long-term suspension culture.

Human bone marrow cells grown in liquid culture in the presence of conditioned medium from concanavalin A stimulated peripheral blood mononuclear cells, from mixed lymphocyte reactions, or from an osteogenic sarcoma tumor line gave rise to basophils which were maximal in number at 1-3 weeks. Basophils had a multilobed nucleus, contained large cytoplasmic granules of variable size which stained metachromatically with acid, but not neutral toluidine blue, were negative for chloroacetate esterase, and did not contain human mast cell tryptase. In long-term cultures, mast cells were detected after 5-6 weeks. Mast cells had cytoplasmic granules that stained with acid and neutral toluidine blue, were positive for chloroacetate esterase, and contained human mast cell tryptase. It, therefore, appears that in liquid cultures even in the absence of feeder layers, human bone marrow cell cultures can give rise initially to basophils, and then to mast cells.     

Generation of purified stromal cell cultures that support lymphoid and myeloid precursors

Treatment of Dexter-type long-term bone marrow cultures with the antibiotic mycophenolic acid (MPA) eliminates all hemopoietic cells from the cultures, while a morphologically intact, adherent stromal cell layer is retained. The ability of these MPA treated stromal cell cultures to support long-term hemopoiesis was tested by seeding them with fresh bone marrow cells that had been passed through nylon wool. This procedure yields a relatively stromal cell depleted population of hemopoietic cells. An aliquot of 5 X 10(5) or 2.5 X 10(5) nylon wool passed bone marrow cells bearing the T6 chromosomal marker was seeded onto replicate MPA-treated stromal cell layers. The stromal cells stimulated the proliferation of the bone marrow cells, and nonadherent cells were present for up to 8 weeks of culture. Progenitors of granulocytes and macrophages (CFU-GM) were also present for this period of time despite weekly demi-depopulation, during culture feeding. Karyotypic analysis confirmed that the CFU-GM were derived from the reseeded population. Nylon wool-passed bone marrow cells seeded alone into empty flasks under identical conditions did not survive past 1 week. Cells from the reseeded cultures were also tested for early myeloid precursors (CFU-S) and injected into immunodeficient CBA/N mice to test for the presence of primitive B cell precursors. CFU-S were present in mice killed 11 days following injection of cells, and high levels of B cell colony-forming units (CFU-B) were present in mice 4 weeks post reconstitution. Further studies demonstrated that factors present in medium conditioned by the stromal cells could support the growth of CFU-GM. These data indicate that treatment of long-term bone marrow cultures with MPA results in a population of functional stromal cells.     

Mast cells in severely T-cell depleted rats and the response to infestation with Nippostrongylus brasiliensis.

The effect of severe T-cell depletion on mucosal mast cells of the small intestine and on connective tissue mast cells has been studied in adult thymectomized, irradiated, bone marrow reconstituted (B) rats. Under normal conditions, intestinal mucosal mast cell numbers do not differ significantly between B rats, normal age matched rats and non-thymectomized irradiated controls. Connective tissue mast cells are significantly fewer in the tongues of B rats than in normal rats, but the difference is atributable to an effect of irradiation. Infestation with Nippostrongylus brasiliensis produced approximately equal increases in mucosal mast cells in non-thymectomized irradiation controls and in normal rats. In B rats there was no increase in mucosal mast cells following infestation. B rats failed to expel the parasites normally. Failure of mast cell proliferation was not due to the effects of the persisting worm burden. Antihelminthic treatment at the time of worm expulsion by normal rats did not reveal a hitherto masked mast cell response in B rats. Nippostrongylus infestation did not reveal evidence of thymus-dependency of connective tissue mast cells. As in athymic nude mice, mucosal mast cells in the rat have been shown to be T-cell dependent during the proliferation that follows infestation with an intestinal nematode parasite.     

Tissue-dependent differences in the asynchronous appearance of mast cells in normal mice and in congenic mast cell-deficient mice after infusion of normal bone marrow cells

The time courses of the appearance of tissue mast cells in six sites were compared in normal WBB6F₁-+/+ mice (+/+) and in congenic mast cell-deficient WBB6F₁-W/W^v mice (W/W^v) that received an intravenous infusion of bone marrow cells from +/+mice (BM→W/W^v). As assessed by morphometric analysis of Carnoy's solution-fixed, methylene blue-stained tissue sections, the density of mast cells in the stomach mucosa, stomach submucosa, and spleen of +/+ mice reached maximal levels by 8 weeks of age, whereas the density of mast cells in the skin, extraparenchymal airway walls, and lung parenchyma did not reach maximal levels until 18 weeks of age. When 8-week-old W/W^v mice were infused with 2×10⁷ bone marrow cells from +/+ mice, mast cells appeared in the stomach mucosa and submucosa after 2.5 weeks, in the spleen and extraparenchymal airway walls after 5 weeks, and in the lung parenchyma after 10 weeks. Twenty weeks after bone marrow infusion, the mast cell densities in the spleen, stomach mucosa, and stomach submucosa were seven-, 13-, and five-fold greater, respectively, than those in age-matched +/+ mice, but were eight-, two-, and five-fold lower in the skin, extraparenchymal airway walls, and lung parenchyma, respectively. Thus, those tissues that in +/+ mice reached maximal mast cell densities earlier exhibited abnormally high mast cell densities in BM→W/W^v mice, and those that reached maximal mast cell densities later in +/+ mice had abnormally low mast cell densities in BM→W/W^v mice. Immunological and inflammatory responses are often compared in W/W^v and BM→W/W^v mice to assess mast cell dependency. Our results indicate that the capacity to restore a mast cell-dependent response in a particular tissue of the latter mice may relate to the local mast cell density and whether the immunological challenge activates mast cells only in that tissue or systematically with attendant widespread release of proinflammatory mediators.     

A simple technique to facilitate the ultrastructural analysis of cells in soft agar culture systems: demonstration of the development in vitro of morphologically mature mast cells and phagocytic macrophages from the bone marrow cells of genetically mast cell-deficient W/Wv or congenic normal mice

We developed a simple technique that greatly facilitates the ultrastructural examination of cells growing in small, widely dispersed colonies in agar medium, and used the method to examine the development of morphologically mature mast cells and actively phagocytic macrophages in agar cultures of mouse bone marrow cells. The bone marrow cells of genetically mast cell-deficient WBB6F1-W/Wv or congenic normal (+/+) mice were cultured in semisolid agar medium supplemented with supernatants of concanavalin A-stimulated splenocytes. To prepare the colonies of hematopoietic cells for transmission electron microscopy, all the colonies within the agar-containing medium in a 96-well culture plate were removed with a Pasteur pipette and placed in a dilute, mixed aldehyde fixative. After fixation, the agar still enmeshing and separating individual colonies of cells was melted at 94 degrees C, rapidly mixed with molten 2% agar in a microfuge tube, centrifuged for 1 minute, and then the plastic tube was cooled in ice for 30 minutes. The plastic was removed with a razor blade, the agar block was hemisected from top to bottom, and then the blocks were processed for electron microscopy, embedded flat, and sectioned for light and electron microscopy. The culture conditions tested resulted in the development of morphologically mature mast cells and actively phagocytic macrophages, whether cultures were initiated with bone marrow cells from WBB6F1-W/Wv or congenic +/+ mice.     

Candida albicans can stimulate stromal cells resulting in enhanced granulopoiesis

Previously, we have reported that although unperturbed granulocyte colony-stimulating factor (GCSF)-deficient (G-CSF-/-) mice are neutropenic, when challenged with Candida albicans, they develop a profound neutrophilia. In an attempt to understand the basis of Candida-induced neutrophilia in G-CSF-deficient mice, we have modified the Dexter bone marrow culture system to produce an in vitro model that mimics emergency granulopoiesis in vivo. In this model, stromal cultures are overlaid with bone marrow cells in the presence or absence of heat-inactivated (HI) Candida. Irrespective of the genotype of mice used as a source of bone marrow-derived stromal cells, stimulation of these cultures with HI Candida led to a significantly greater recovery of cells compared to unstimulated stromal cultures. In addition, there was a marked increase in the number of colony-forming units granulocyte-macrophage (CFU-GM), as well as in the percentage of granulocytes in the population of nonadherent cells recovered from HI Candida-stimulated cultures. The conditioned medium generated from stromal cultures derived from either wild-type or G-CSF-/- mice exposed to HI Candida, when applied to bone marrow cells in a soft agar clonogenic assay stimulated M-, GM-, and G- type colonies. Interleukin-3 (IL-3) and GM-CSF could not be detected in the conditioned medium from either HI Candida stimulated or unstimulated stromal cultures. However, IL-6 was detected in the conditioned media from both wild-type and G-CSF-/- stromal cultures. Addition of anti-IL-6 antibody significantly impaired granulopoiesis in unstimulated and HI Candida-stimulated, wild type, and G-CSF-/- stromal cultures. Conditioned medium generated from G-CSF/IL-6-deficient stromal cells had the capacity to stimulate bone marrow cells to form colonies comprised of granulocytes and macrophages in soft agar clonogenic assay. This study demonstrates that stromal cells can be stimulated with HI Candida and gives an insight into Candida mediated granulopoiesis.     

Light-chain diversification during pre-B cell differentiation

The possibility that a committed normal pre-B cell can generate progeny expressing more than one light chain was studied by isoelectric focusing of supernatants from pre-B cells cultured at limiting dilution. Supernatants from mitogen-stimulated mature spleen B-cells analysed on day 6 have shown the presence of a rather homogeneous IgM spectrotype profile. Supernatants from the 8–10-day cultures were usually negative. Isoelectric focusing profiles of supernatants from cultures of bone marrow pre-B cells were different from those of mature spleen cells. Many IgM spectrotypes appeared in the culture supernatants of bone marrow pre-B cells between days 6 and 13, whether the cultures were negative or restricted in their IgM profiles on day 6. These results support the idea that normal pre-B cells, during differentiation, can associate a variety of light chains with an already chosen μ heavy chain.     

Interleukin-4-triggered, STAT6-dependent production of a factor that induces mouse mast cell apoptosis

IL-4 can suppress mast cell development from mouse spleen, bone marrow and peritoneal cells by an indirect process that is dependent on the presence of macrophages. Mast cells undergo apoptosis when exposed to supernatants collected from cultures of IL-4-stimulated peritoneal cells due to the IL-4-induced production of an apoptosis-inducing factor in the cultures. This effect of IL-4 is shown to be dependent on STAT6 signaling, because IL-4 and IL-13 do not suppress mast cell development from the spleen and peritoneal cells of STAT6-/- mice. Moreover, supernatants from cultures of IL-4- and IL-13-stimulated peritoneal cells of STAT6-/- mice do not exhibit apoptosis-inducing activity. We confirm, by using deficient mice, neutralizing antibodies and recombinant cytokines, that IL-4-induced apoptosis is not related to the well-known apoptosis-inducing factors Fas, Fas ligand, TNF-alpha, TRAIL, TGF-beta or perforin. These results demonstrate a novel mechanism whereby IL-4 and IL-13 can suppress mast cell development by inducing the production of an apoptosis-inducing factor from macrophages.     

Antigen-induced proliferation in vitro of bone marrow precursors of granulocytes and macrophages

The injection of polymerized flagellin into C57BL mice increased the number of granulocyte cluster-forming cells and macrophage colony-forming cells in the bone marrow. The addition of polymerized flagellin to agar cultures of bone marrow cells from normal or antigen-injected mice increased the number and growth rate of macrophage colonies developing when mouse serum was used as the source of colony stimulating factor (CSF). Polymerized flagellin had no potentiating effect on colony growth when urine or medium conditioned by bone marrow, spleen, lymph node or thymic cells were used as the source of CSF. Antigens may stimulate the proliferation of granulocyte and macrophage precursor cells by an indirect mechanism involving the production by bone marrow cells of CSF, or a similar growth promoting factor.     

Effect of a Trichinella spiralis infection on the distribution of mast cell precursors in tissues of thymus-bearing and non-thymus-bearing (nude) mice determined by an in vitro assay.

The frequency of precursor cells capable of giving rise to cells with characteristics of mucosal mast cells in tissues from thymus-bearing and non-thymus-bearing (nude) mice orally infected with Trichinella spiralis was determined with an in vitro assay. Analysis of the frequency of mast cell precursors in bone marrow, blood, spleen and small intestinal tissue revealed similar frequencies of mast cell precursors in bone marrow from both thymus-bearing and athymic mice. These frequencies in bone marrow were not affected by infection. However, in blood and spleen from thymus-bearing mice at Day 7 post-infection (p.i.), and in the gut at Day 14 p.i., significant increases of mast cell precursor frequencies were detected. In contrast, no significant increase was observed in the tissues of infected nude mice. These data are in accordance with in vivo findings, indicating that a mucosal mast cell response in the gut is both thymus and antigen dependent. It was concluded that a mucosal mast cell response to infection with T. spiralis is probably due to local proliferation and maturation of residing mast cell precursors, that this response might be amplified by an influx of precursor cells from the blood into the gut, and that both phenomena are T-cell dependent.     

急性髓系白血病干细胞NOD/SCID小鼠白血病模型的建立

目的探讨用非肥胖型糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠和经流式细胞仪分选出的KG1a中CD34＋CD38-的干细胞亚群建立白血病干细胞（LSCs）动物模型,为靶向治疗LSCs的药物筛选奠定体内实验基础。方法 18只雌性NOD/SCID小鼠,体质量18～20 g,鼠龄6～8周龄。实验组12只,应用流式细胞仪分选KG1a细胞中具有LSCs特性的CD34＋CD38-亚群,以2×106个/只尾静脉注射经全身X射线照射2Gy的NOD/SCID小鼠;正常对照组6只,只注射磷酸盐缓冲溶液。观察两组小鼠的一般情况和白血病发生情况,应用形态学、组织病理检查、流式细胞术、骨髓染色体检查等检测实验组小鼠的外周血、骨髓、肝脏、脾脏的白血病细胞标志。结果接种2周后实验组小鼠外周血可见白血病细胞,接种30 d实验组小鼠的白血病发病率为100%,无自发缓解;外周血、骨髓、肝、脾中均可发现大量的白血病细胞浸润;实验组小鼠骨髓细胞中CD13抗原阳性率15.47%～23.66%,并可见KG1a细胞的核型特征。结论尾静脉接种流式细胞仪分选后的CD34＋CD38-KG1a细胞于全身亚致死量X射线照射后的NOD/SCID小鼠,能成功建成全身播散的白血病模型,为进一步研究LSCs的靶向治疗药物奠定实验基础。     

Precursor cells of fibroblasts detected by in vitro cloning of cells from hematopoietic organs of normal and irradiated mice

Colonies of fibroblasts are formed in monolayer cultures of bone marrow, spleen, and thymus cells of adult mice with an efficiency of colony formation (per 10⁵ cells) of 2.2 for bone marrow, 0.20 for spleen, and 0.16 for thymus. On irradiation of mice with a dose of 150 R, about half of the fibroblasts colony-forming units in the bone marrow die; during the next 6 days their number falls a further fivefold, with a return to the normal level 25 days after irradiation.Laboratory of Immunomorphology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 614–617, May, 1976.     

Functional Thy-1+ cells in cultures of spleen cells from nu/nu mice

Thy-1⁺ lymphocytes, detectable by quantitative serum absorption, arise in cultures of spleen cells from congenitally athymic (nu/nu) mice supplemented with supernatants from cultures of normal spleen cells stimulated with the T cell mitogen concanavalin A. Pretreatment of nu/nu spleen cells with appropriate anti-Thy-1 alloantibody and complement prior to culture reduces their capacity to generate Thy-1⁺ cells by about 90%. This shows that the majority of cells proliferating in these cultures are descendants of Thy-1⁺ cells which can be detected in the original nu/nu spleens. Experiments aimed at exploring the function of these Thy-1⁺ cells after culture in conditioned medium revealed that within one or two days after culture initiation, strong cooperative activity for a humoral response to xenogeneic erythrocytes can be detected. In mixtures of bone marrow-derived lymphocytes from various mouse strains with cultured nu/nu spleen cells, it was observed that T-B cooperation is not H-2-restricted. Attempts at inducing T cell-mediated cytotoxicity to alloantigens in such cultures of nu/nu spleen cells were unsuccessful. In contrast, nonspecific cytotoxicity which was attributed to natural killer cells was regularly observed and could be maintained in these cultures over extended periods.     

Factors affecting the growth and maintenance of human skin mast cells in cell culture.

The isolation and kinetics of survival of human mast cells from newborn and adult skin is described. Recombinant human interleukins and conditioned medium from several human cell lines were tested for their ability to maintain mast cells in vitro. Growth medium supplemented with IL-2, IL-4 and conditioned medium from a mixed lymphocyte culture enhanced mast cell survival resulting in a 30-fold increase in survival (relative to that obtained with non-supplemented medium) at 7 days, and a 15-fold increase at 15 days. Cell survival for time periods longer than 21 days was not observed. Inclusion of cAMP, agents that elevate cAMP, insulin, and epidermal growth factor in supplemented growth medium prevented the enhanced survival by 40-70%. Incorporation of bromodeoxyuridine (BrdU) into mast cells in 3-day cultures demonstrated that 15% of the mast cell population was capable of proliferation. At 21 days, no incorporation of BrdU could be detected. After 3 days in culture mast cells released 16% of their histamine stores in response to A23187 and 10% in response to anti-human IgE. Electron microscopy of cultured cells at 3 days revealed cells with both intact and empty mast cell granules. These results demonstrate that human skin mast cells proliferate in response to cytokines and release histamine when stimulated with classical secretagogues. Since human skin mast cells retain these basic properties in vitro, they may be useful in further functional studies involving their proliferation and secretion.     

Development and maturation of Langerhans cells, spleen and bone marrow dendritic cells in TNF-alpha/lymphotoxin-alpha double-deficient mice

Dendritic cells are key regulators of immunity and tolerance. TNF-alpha has manifold effects on dendritic cells. It is an indispensable ingredient in several dendritic cell generation protocols, especially in the human, and it is included in diverse maturation stimuli for dendritic cells. Mice deficient in various components of the TNF/lymphotoxin system (TNF-alpha, lymphotoxin-alpha and -beta, TNF receptors, combinations thereof) have profound defects in mounting immune responses to infections. The dendritic cell system in these mice has been incompletely studied to date. We therefore investigated dendritic cells from the epidermis (Langerhans cells), spleen and the bone marrow of mice double-deficient in TNF-alpha and lymphotoxin-alpha. We report that dendritic cells in these mice are grossly normal. Langerhans cells, spleen and bone marrow dendritic cells can develop and mature. Their expression of MHC II and CD86 is not impaired, and their T cell-stimulatory as well as antigen-processing capacity is comparable to their normal counterparts. Thus, the described defects in these mice appear to be due the lack of lymph nodes, the disturbed architecture of the spleen, and deranged chemokine production patterns, rather than to a profoundly altered dendritic cell system.