New monoclonal antibody that specifically recognizes murine interdigitating and Langerhans cells

A new monoclonal antibody, M1-8, that recognizes murine interdigitating cells (IDC) and Langerhans cells was obtained from a hybridoma prepared by fusion of SP2/0 mouse myeloma cells with splenic cells of rats immunized with IDC-rich cell suspension obtained from lymph nodes of athymic nude mice (BALB/c nu/nu). The specificity was assessed immunohistochemically on frozen sections of lymph nodes and epidermal sheets from both nude and normal mice. M1-8 reacted with paracortical IDC, veiled cells of the marginal sinus, and epidermal Langerhans cells in both normal and nude mice. In simultaneous staining by M1-8 and nonspecific esterase or anti-Ia or anti-Thy-1,2 antibody, the same epidermal dendritic cells were positive for all these antigens except Thy-1,2. Immunoelectron microscopy of the lymph node suspension using gold colloid particles revealed the attachment of gold particles to the cell membrane of IDC. Analysis by flow cytometry of the lymph node cell suspension showed 14 or 6% of M1-8-positive cells in nude or normal mouse, respectively. Immunohistochemical analysis showed that M1-8 also reacted with dendritic cells in the thymus and spleen and had a different distribution from F4/80. M1-8 also reacted with monocytes in bone marrow and peripheral blood, alveolar macrophages, and thioglycollate-stimulated peritoneal exudate macrophages. The antibody belongs to the immunoglobulin M class, reacts immunochemically with a glycoprotein in the cell membrane, and has a molecular mass of approximately 15 kDa.     

Abnormal development and differentiation of macrophages and dendritic cells in viable motheaten mutant mice deficient in haematopoietic cell phosphatase

In mice homozygous for the 'viable motheaten' ( me^v ) mutation, numbers of macrophage progenitor cells, particularly monocytes, were markedly increased in the bone marrow and spleen. Increased mobilization of these precursor cells to peripheral tissues and their differentiation to macrophages were evidenced by striking increases in macrophage numbers. Immunohistochemical double staining of tissue sections and flow cytometry analyses of single cell suspensions from these mice demonstrated CD5 (Ly-1)-positive macrophages in the peritoneal cavity, spleen and other tissues. Ly-1-positive macrophage precursor cells were demonstrated in the peritoneal cavity of the me^v mice and developed in the omental milky spots. The development of marginal metallophilic and marginal zone macrophages was poor in the splenic white pulp and related macrophage populations were absent in the other lymphoid tissues. The numbers of epidermal Langerhans cells in the skin and T cell-associated dendritic cells in the thymic medulla, lymph nodes, and the other peripheral lymphoid tissues were decreased. However, increased numbers of dendritic cells accumulated in the lungs, liver, and kidneys. These abnormalities in development and differentiation of macrophages and dendritic cells may be ascribed to the deficiency in haematopoietic cell SHP-1 tyrosine phosphatase or may be a secondary consequence of abnormal microenvironments, (either constitutive or in response to inflammatory stimuli) in the haematopoietic and lymphopoietic organs and tissues of these mice.     

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.     

Sarcoma of follicular dendritic cells with features of sinus lining cells—a new subtype of reticulum cell sarcoma?

Dendritic cell neoplasms of the World Health Organization classification comprise Langerhans cell histiocytosis, Langerhans cell sarcoma, interdigitating dendritic cell sarcoma, follicular dendritic cell sarcoma, and dendritic cell sarcoma, not otherwise specified. Several studies based on immunohistochemical and ultrastructural analysis tried to further clarify the origin of these neoplasms which are thought to derive from mesenchymal or bone marrow precursors. Lymphatic vessel endothelium hyaluronan receptor-1 (LYVE-1) was recently described as a marker for lymphatic endothelium which is expressed on normal liver blood sinusoid lining cells, spleen endothelium, activated tissue macrophages, blood vessels in the lung, endothelial cells of lymphatic sinuses, and in fibroblastic reticular cells in lymph nodes. We present a case of LYVE-1-positive reticulum cell neoplasm in an axillary lymph node. To the best of our knowledge, there has been no report about LYVE-1 expression in histiocytic or dendritic cell neoplasms so far. Due to the assumed specificity of this antibody, we propose designation of this reticulum cell sarcoma as lymphatic sinus lining cell sarcoma which might finally represent another subtype of reticulum cell sarcomas.     

Skewed differentiation of bone marrow CD34+ cells of tumor bearers from dendritic toward monocytic cells,and the redirection of differentiation toward dendritic cells by 1alpha,25-dihydroxyvitamin D3

Tumor presence is detrimental to the development of antigen-presenting dendritic cells. Since dendritic cells can arise from CD34+ precursor cells, the present study assessed the capacity of bone marrow CD34+ cells from tumor bearers to develop into dendritic cells when cultured in the absence of either tumor cells or their products. Culturing bone marrow CD34+ cells from mice bearing Lewis lung carcinomas yielded a lower number of dendritic cells than arose from CD34+ cells of normal mice. This reduced yield of dendritic cells was associated with a shift to development of monocytic cells and a reduced antigen presenting capability by the cultures. When the CD34+ cell cultures from tumor bearers were supplemented with the differentiation-inducing hormone 1alpha,25-dihydroxyvitamin D3, there was the restoration of dendritic cell development and antigen presenting ability. These results show that CD34+ cells from tumor bearers remain defective in their development into dendritic cells even when cultured outside the tumor environment, but development of dendritic cells can be restored with 1alpha,25-dihydroxyvitamin D3.     

Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells.

The bulk of natural IgM secretion is currently attributed to peritoneal CD5+ B cells and their progeny, believed to be independent of adult bone marrow precursors. We have compared the capacity of peritoneal or splenic cells from normal adult mice to generate serum IgM after transfer into allotype-congenic, irradiated and bone marrow-protected mice. Recipients of either cell population produced donor-allotype IgM-secreting cells in the spleen, and had donor-derived serum IgM. In both cases as well, recipient IgM secretion recovered to control levels. Since the spleen cell-derived natural IgM production could result from expansion of CD5+ B cells present in the inoculum, we next investigated the ability of Ig- bone marrow (BM) cells (Ig- BM) to reconstitute natural IgM secretion in irradiated mice. This cell population was most efficient in reconstituting donor-derived IgM secretion. The origin and phenotype (IgM, CD5) of B cells present in spleen and peritoneum of recipient mice were also analyzed. In agreement with the high level of donor IgM-secreting cells, transfers of splenic and Ig- BM cells fully reconstitute donor B cells in spleen and peritoneum and inhibit reconstitution from host origin. In contrast, donor peritoneal cells reconstitute B cells very poorly in spleen and allow for reconstitution by host cells. Furthermore, Ig- BM cells as well as splenic or peritoneal donor cells, all reconstitute CD5+ B cells in the peritoneum of recipient mice. Interestingly, the fraction of IgM+ cells of each allotype that differentiate to IgM secretion varies widely, but normal levels of IgM are established even when the number of donor B cells present in the animal is very limited.     

Effects of TNF-alpha inhibitors on the number of epidermal Langerhans cells in uninvolved skin of psoriatic patients: A pilot study

Only limited data are available on the effects of TNF-alpha inhibitors on dendritic cells. However, TNF-alpha plays a central role in the biology of dendritic cells, both with regard to their maturity process and mobilization to secondary lymphoid organs. In particular, the effects of TNF-alpha inhibitors on Langerhans cells in healthy skin have never been investigated. In this pilot study, we aimed to assess the change of the density of Langerhans cells within the normal, not photo-exposed, skin of 17 psoriatic patients, before and after 16 weeks of treatment with TNF-alpha inhibitors. Most of the patients (88%) showed an increase or a similar density of Langerhans cells after 16 weeks of therapy with TNF-alpha inhibitors compared with baseline values. Only 2 patients (12%) showed a reduction of these cells following therapy with TNF-alpha inhibitors.     

Gene profiling approach in the analysis of lymphotoxin and TNF deficiencies

Mice with combined lymphotoxin-alpha (LTalpha) and tumor necrosis factor (TNF) deficiencies show defects in the structure of peripheral lymphoid organs such as spleen, lymph nodes, and gut-associated lymphoid tissues. To identify genes associated with this defective phenotype in spleen, we applied a gene profiling approach, including subtractive cloning and gene array hybridizations, to mice with combined TNF/LT deficiency. The differentially expressed genes identified by these techniques was then evaluated by Northern blot analysis for splenic expression in knockout mice with single LTalpha or single TNF deficiency. Most of the genes detected in this analysis are directly or indirectly associated with disrupted LT and not TNF signaling.     

Migration of langerhans cells and dermal dendritic cells in skin organ cultures: augmentation by TNF-alpha and IL-1beta.

Migration from sites of antigen encounter to lymphoid organs is essential to the strong immunogenic function of dendritic cells (DC). In the skin, migration proceeds through dermal lymphatic vessels and is regulated in an incompletely understood way by inflammatory mediators. We studied the effects of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in mouse skin organ cultures by direct enumeration of migrating DC and by immunohistochemistry. (1) Neutralizing antibodies to TNF-alpha and IL-1beta inhibited migration of DC, also in human skin explants (TNF-alpha). (2) TNF-alpha at low concentrations (50 U/mL) and IL-1beta (50-3000 U/mL) augmented migration to about 150% of spontaneous migration. (3) High concentrations of TNF-alpha (5000 U/mL) inhibited migration by approximately 50%. (4) DC migration from skin explants of TNF-alpha/lymphotoxin-alpha double-deficient mice and TNF-receptor type 1 and 2 double knockout mice was not impaired. (5) TNF-alpha effects were neutralized by anti-IL-1beta, and vice versa. We conclude that in normal animals both TNF-alpha and IL-1beta are required for DC migration to occur. In the complete absence of one cytokine (TNF-alpha), however, backup mechanisms step in.     

T lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin II (Prx II) gene

Peroxiredoxin II (Prx II) is a member of antioxidant enzyme family and it plays a protective role against oxidative damage. Constitutive production of endogenous reactive oxygen species was detected in spleen and bone marrow cells lacking Prx II. Here, we investigated the role of Prx II in immune responses. The total number of splenocytes (especially, the population of S-phase cells and CD3(+) T cells) was significantly higher in Prx II(-/-) mice than in wild type. Number of peripheral blood mononuclear cells (PBMCs) in Prx II(-/-) mice was also higher than wild type. Differentiation of Prx II(-/-) mouse bone marrow cells into CD11c-positive dendritic cells was greater than that of wild type. Transplantation of Prx II(-/-) bone marrow cells into wild type mice increased PBMCs in blood and bone marrow-derived dendritic cells. Prx II deletion enhances concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction (MLR) activity of bone marrow-derived CD11c-positive dendritic cells to stimulate recipient splenocytes. Collectively, these data suggest that Prx II inhibits the immune cell responsiveness, which may be regulated by scavenging the low amount of reactive oxygen species (ROS).     

黄芪多糖对小鼠骨髓及外周血造血干细胞的增殖及动员作用

探讨APS-P对正常或化疗后小鼠的骨髓、脾及外周血造血干细胞的促增殖及外周动员作用。给正常或经环磷酰胺化疗后的C57BL／6小鼠注射APS-P，然后取骨髓细胞、外周血细胞及脾细胞，并用荧光抗体PerCP-Sca-1，APE-c-kit及PE-Lineage(CD3，CD4，CD8，Trll9，B220 CDllb，Gr-1)进行染色标记，用流式细胞仪检测小鼠造血干细胞(Lin c-kit^ Sca-1^ )数量变化。注射APS-P能使正常及化疗小鼠骨髓、脾及外周血中的造血干细胞有不同程度的增加。这一作用可能是由于APS-P促进骨髓造血干细胞的增殖，而在外周血及脾中的增加则可能是由于APS-P对骨髓造血干细胞的外周动员作用。     

Alterations in the phenotype and function of immune cells in ovariectomy-induced osteopenic mice

BACKGROUND: Within the last few years, much evidence has been presented on the involvement of the immune system in certain types of bone loss, such as activated T cells in rheumatoid arthritis and in periodontitis. Estrogen deficiency induces bone loss; however, how this deficiency affects the immune system has not been sufficiently studied. METHODS: To evaluate the effects of estrogen withdrawal on the status and functionality of the immune system, mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analysed in spleen and in bone marrow. We analysed bone turnover, cell phenotype by flow cytometry, cell function by cell proliferation assays, and the expression of several genes related to the process. RESULTS: Five weeks after ovariectomy, augmented osteoclastogenesis persisted in the bone marrow. In addition, the ovariectomized mice had more B-cells and CD3+ T-cells expressing the receptor activator of NF-kappaB ligand (CD3+/RANKL+). The ovariectomized mice had lower serum alkaline phosphatase activity, a normal amount of T cells, lower percentages of CD11b+ and CD51+ cells in the bone marrow, and a lower serum interferon-gamma level compared with sham-operated controls. CONCLUSIONS: The data suggest that, 5 weeks after ovariectomy, bone turnover remains imbalanced, with increased osteoclastogenesis and a decreased rate of bone formation. Moreover, there is an increase in B-cell formation, with normal and decreased percentages of T cells and myelomonocytic cells (CD11b+), respectively, in the bone marrow. Decreased serum interferon-gamma levels could be involved in the increased osteoclastogenesis found in the present work.     

Medium conditioned by spleen cells of Nematospiroides dubius-infected mice does not support development of cultured mast cells

The ability of different conditioned media to support mast cell development from precursors in normal bone marrow was evaluated. Many mast cells developed in bone marrow cultures containing medium conditioned by concanavalin-A-stimulated spleen cells of normal mice, Trichinella spiralis-infected mice, or mice infected for 6 days by Nematospiroides dubius. By contrast, medium conditioned by concanavalin-A-stimulated spleen cells of mice having 18-day infections of the nematode N. dubius failed to support mast cell development. The difference between medium conditioned by spleen cells of mice having 6-day versus 18-day infections of N. dubius may be due to the life cycle stage of the parasite, larval or adult, present at those times. These results from cultures are consistent with those from in vivo studies in which mice given primary infections of N. dubius failed to develop the intestinal mastocytosis characteristic of nematode infections.     

CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells

CD45 is known to regulate signalling through many different surface receptors in diverse haemopoietic cell types. Here we report for the first time that CD45-/- bone marrow dendritic cells (BMDC) are more activated than CD45+/+ cells and that tumour necrosis factor (TNF) and interleukin-6 (IL-6) production by BMDC and splenic dendritic cells (sDC), is increased following stimulation via Toll-like receptor (TLR)3 and TLR9. Nuclear factor-kappaB activation, an important downstream consequence of TLR3 and TLR9 signalling, is also increased in CD45-/- BMDC. BMDC of CD45-/- mice also produce more TNF and IL-6 following stimulation with the cytokines TNF and interferon-alpha. These results show that TLR signalling is increased in CD45-/- dendritic cells and imply that CD45 is a negative regulator of TLR and cytokine receptor signalling in dendritic cells.     

Maturation of antigen-presenting cells is compromised in HLA-G transgenic mice

The human MHC class Ib antigen HLA-G is thought to regulate maternal immune responses during pregnancy. Here we show that expression of HLA-G in transgenic mice diminished cellular immunity by inhibiting maturation of myelomonocytic cells into functional antigen-presenting cells (APC). Skin allografts applied to HLA-G transgenic mice survived longer and resultant T cell responses were less potent compared to control mice. T cells from HLA-G mice responded normally to allogeneic APC and immunohistological analyses of spleen revealed no marked abnormalities. However, spontaneous outgrowths of myeloid cells were observed when bone marrow or splenocytes from HLA-G mice were cultured in vitro, but functionally competent APC did not develop spontaneously in bone marrow cultures supplemented with granulocyte macrophage colony stimulating factor (GM-CSF). Addition of lipopolysaccharide (LPS) to GM-CSF-derived bone marrow cultures rescued APC maturation. Studies using HLA-G tetrameric reagents revealed that HLA-G-specific binding activity was associated with CD11c(+) myelomonocytic cells, while binding to lymphoid and NK cell subsets was undetectable. These data show that spontaneous maturation of functionally competent dendritic cells (DC) is compromised in HLA-G mice. We hypothesize that HLA-G inhibits maturation of DC via receptor-mediated interactions with myelomonocytic precursors, which render immature DC precursors unable to receive signals from activated T cells.     

Tumor regression by anti-CD40 and interleukin-2: role of CD40 in hematopoietic cells and organ-specific effects.

CD40 stimulation can synergize with interleukin (IL)-2 for antitumor responses against mouse metastatic renal cell carcinomas, with coincident increases in tumor-specific CD8+ T-cell responses and dendritic cell numbers in both the spleen and liver. Because CD40 is present on various hematopoietic-derived cells, endothelial cells, and some tumors themselves, this study was performed to determine whether the antitumor effects of CD40 stimulation and IL-2 were primarily mediated by CD40+ hematopoietic-derived cells. Bone marrow chimeras were created by reconstituting lethally irradiated CD40+/+ recipients with bone marrow from CD40-/- or CD40+/+ mice. Chimeric mice were then implanted orthotopically with renal cancer cells, followed by treatment with anti-CD40 agonist monoclonal antibodies and IL-2. Immune parameters of the spleen and liver were assessed after therapy and correlated with antitumor responses. The antitumor effects in the CD40-/- bone marrow transplantation chimeras were almost completely abrogated after treatment, and this shows that hematopoietically derived CD40+ cells are the principal targets for CD40 stimulation in this model. Although both spleen and liver showed reductions in CD8+ T-cell and dendritic cell expansion in the CD40-/- versus CD40+/+ chimeras after therapy, only the liver exhibited no significant increases in either CD8+ T cells or dendritic cells after treatment. CD40 cells on hematopoietic cells are the primary target for anti-CD40 and IL-2 therapy. The results also suggest that the immunologic events in the liver may be more revealing that those in lymphoid organs with regard to critical events related to responses after therapy.     

Macrophages,but not dendritic cells,present collagen to T cells

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen α chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.     

Low responsiveness of hepatitis B virus-transgenic mice in antibody response to T-cell-dependent antigen: defect in antigen-presenting activity of dendritic cells.

The experiments presented here were performed to evaluate immune responsiveness of hepatitis B virus (HBV)-transgenic mice (transgenic mice), as a model of HBV-carrier state to a T-cell-dependent antigen, keyhole limpet haemocyanin (KLH). The transgenic mice which were completely unresponsive to hepatitis B surface antigen (HBsAg), responded poorly to KLH. The levels of anti-KLH antibodies (Ab) produced in vivo were significantly lower in transgenic mice compared with the normal control mice at respective immunizing doses of KLH. In addition, a little or no anti-KLH Ab production was detected in culture supernatants of KLH-primed transgenic mice spleen cells. KLH-primed T cells from normal and transgenic mice induced anti-KLH Ab production from transgenic B cells in the presence of antigen-presenting spleen adherent cells (SAC) from normal mice, but not those from transgenic mice. Depletion of dendritic cells from normal mice-derived SAC completely abrogated the anti-KLH Ab response in transgenic spleen cell culture and their addition to the culture restored the response. Low efficiency of transgenic dendritic cells was demonstrated in sodium periodate (NaIO4)-induced non-specific and allogenic antigen-induced T-cell proliferation. Finally, cytofluorometric analyses showed a reduced Ia antigen expression on transgenic dendritic cells. These results indicate that low responsiveness of transgenic mice in specific-antibody response is not due to functional defects in T cells or B cells but rather to a defect of antigen-presenting activity of dendritic cells.     

A comparison of baseline and cyclophosphamide-induced sister chromatid exchanges in bone marrow and spleen cells of mouse and Chinese hamster

Baseline sister chromatid exchange (SCE) frequencies were investigated in bone marrow and spleen cells of mice and Chinese hamsters. No significant difference in SCE frequency was noted for bone marrow in both species and for bone marrow and spleen in mice on per cell and per pg DNA basis. However, a significant difference was noted between species in spleen and between cell types in Chinese hamsters. Also, statistically significant differences were noted between species for both cell types when the same data were expressed on per chromosome basis. SCE levels in cultured bone marrow and spleen cells after intraperitoneal administration of the antineoplastic drug cyclophosphamide (10 and 20 mg/kg) differed significantly in mice and Chinese hamsters on per cell, per pg DNA content, and per chromosome basis. The spleen cells were much more sensitive to the effects of cyclophosphamide than bone marrow cells in both species. The replicative indices did not differ significantly between treated and control animals in either bone marrow or spleen cells of both species. Since SCE frequency is a sensitive measure of DNA damage, and bone marrow and lymphocytes are the most widely used cell types in human and animal in vivo assays, the methodologies and results reported here may be useful for comparative mammalian cytogenetic studies.