Early appearance of functional plasma cells (CD138+CD98+) in non-immunized neonate mice.

Plasma cells (PCs) are terminally differentiated antibody-secreting cells, derived from activated B-lymphocytes in response to either T-independent or T-dependent antigens. The plasma cell population is scarce in circulation in non-immunized individuals. It is established that neonates are incapable of mounting an efficient immune response due to the immaturity of the immune system. However, this disadvantage is well overcome through the antibodies neonates receive from breastmilk. This implies that neonates will be only protected against antigens the mother had previously encountered. Thus, the child might be potentially susceptible to new antigens. This issue prompted us to seek for the presence of PCs in non-immunized neonate mice. We found a PC population identified as CD138+/CD98+ cells since day one after birth. These PCs were positive for Ki67 and expressed Blimp-1, B220, and CD19, which suggests the populations are plasmablasts and PCs with heterogeneous phenotype. These PCs were also determined to secrete antibodies, although mainly isotype IgM. Altogether, the results indicated that neonate PCs can produce antibodies against antigens they encounter in the first weeks of life, most likely coming from food, colonizing microbiota, or the environment.

Monocyte Locomotion Inhibitory Factor confers neuroprotection and prevents the development of murine cerebral malaria.

Cerebral malaria (CM) is a neurological complication derived from the Plasmodium falciparum infection in humans. The mechanisms involved in the disease progression are still not fully understood, but both the sequestration of infected red blood cells (iRBC) and leukocytes and an exacerbated host inflammatory immune response are significant factors. In this study, we investigated the effect of Monocyte Locomotion Inhibitory Factor (MLIF), an anti-inflammatory peptide, in a well-characterized murine model of CM. Our data showed that the administration of MLIF increased the survival and avoided the neurological signs of CM in Plasmodium berghei ANKA (PbA) infected C57BL/6 mice. MLIF administration down-regulated systemic inflammatory mediators such as IFN-γ, TNF-α, IL-6, CXCL2, and CCL2, as well as the in situ expression of TNF-α in the brain. In the same way, MLIF reduced the expression of CD31, CD36, CD54, and CD106 in the cerebral endothelium of infected animals and prevented the sequestration of iRBC and leucocytes in the brain microvasculature. Furthermore, MLIF inhibited the activation of astrocytes and microglia and preserved the integrity of the blood-brain barrier (BBB). In conclusion, our results demonstrated that the administration of MLIF increased survival and conferred neuroprotection by decreasing neuroinflammation in murine CM.

A CCL chemokine-derived peptide (CDIP-2) exerts anti-inflammatory activity via CCR1, CCR2 and CCR3 chemokine receptors: Implications as a potential therapeutic treatment of asthma.

Allergic asthma is a chronic inflammatory disease characterized by the accumulation of eosinophils, Th2 cells and mononuclear cells in the airways, leading to changes in lung architecture and subsequently reduced respiratory function. We have previously demonstrated that CDIP-2, a chemokine derived peptide, reduced in vitro chemotaxis and decreased cellular infiltration in a murine model of allergic airway inflammation. However, the mechanisms involved in this process have not been identified yet. Now, we found that CDIP-2 reduces chemokine-mediated functions via interactions with CCR1, CCR2 and CCR3. Moreover, using bone marrow-derived eosinophils, we demonstrated that CDIP-2 modifies the calcium fluxes induced by CCL11 and down-modulated CCR3 expression. Finally, CDIP-2 treatment in a murine model of OVA-induced allergic airway inflammation reduced leukocyte recruitment and decreases production of cytokines. These data suggest that chemokine-derived peptides represent new therapeutic tools to generate more effective antiinflammatory drugs.

Characterisation of the green turtle's leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity.

Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27-38 %; 39-45 %) and OVA-Alexa (35-46 %; 14-22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis.

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.

T-cell responses and in vivo cytotoxicity in the target organ and the regional lymphoid tissue during airborne infection with the virulent Mycobacterium tuberculosis MT103 and its lipid mutant fadD26.

To ascertain the in vivo role of mycobacterial lipids phthiocerol dimycocerosates (PDIM) in experimental murine tuberculosis (Tb), airways infection was used to compare the parental virulent clinical isolate MT103 with its mutant fadD26, lacking PDIM. Lungs were assessed as the Tb-target organ and mediastinal lymph nodes as the corresponding lymphoid tissue, in order to quantify: the major T-cell subsets (CD4+/CD8+/gammadelta+) and their activation kinetics, bacillary burden, and in vivo cytotoxicity against inoculated target cells loaded with mycobacterial Ags. After 4 weeks, infection augmented total and activated CD4+ and CD8+ T cells in lungs and nodes mainly with MT103, while gammadelta+ T cells increased earlier in nodes. MT103 bacillary burden was bigger and appeared earlier than the mutant fadD26, especially in the lung than in mediastinal nodes. At day 14 of MT103 infection, there was no cytotoxicity in lungs and nodes; while with fadD26 there was some in the nodes. At day 21 of MT103 infection, important cytotoxicity was detected only in lungs; while with fadD26 both tissues showed important activity. Interestingly, unlike the infection with fadD26, cytotoxicity under MT103 fell considerably in the target organ (lung) from days 21 to 60, the advanced phase. Although upon airways infection both mycobacteria behaved similarly regarding T cell (CD4/CD8/gammadelta) stimulation kinetics; they differed in the magnitude of these responses, in the bacterial load within tissues, and to trigger in vivo cytotoxicity in lungs and regional lymph nodes. This highlights the relevance of certain mycobacterial lipids to modify crucial effector branches of immunity.

Rational design of synthetic peptides to generate antibodies that recognize in situ CD11c(+) putative dendritic cells in horse lymph nodes.

A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in peripheral blood cell suspensions and in cryosections of horse lymph nodes. Only the serum against peptide 2 was capable of identifying cells in suspension and in situ by immunohistochemistry, some with evident dendritic morphology. Using this approach, an immunogenic epitope exposed in CD11c was identified in cells from horse lymph node in situ.

Epidermal Langerhans cells in Actinic Prurigo: a comparison between lesional and non-lesional skin.

BACKGROUND: Actinic Prurigo (AP) is a chronic pruritic dermatosis of unknown cause affecting sun exposed skin in defined ethnic groups with characteristic MHC alleles. However, the cutaneous dendritic cells have not been assessed. OBJECTIVE: To assess in situ the epidermal Langerhans Cell (LC) status in Actinic Prurigo. STUDY DESIGN: Fresh skin samples from three AP patients were used to evaluate in situ the epidermal LC, comparing lesional and non-lesional sites in each subject. SETTING: AP patients attending the Dermatology Department at the Hospital M. Gea-Gonzalez in Mexico city. METHODS: Lesional and non-lesional skin samples were taken from each subject to prepare both epidermal sheets and conventional tissue sections. Three markers restricted to LC in epidermis (CD1a, ATPase, MHC-II) were used to quantify the LC per area in epidermal sheets. RESULTS: Compared to non-lesional skin from the same subject, a significant reduction in the number of LC per area of epidermis was found in lesional skin; with any of the three markers evaluated. CONCLUSION: The frequency of epidermal LC decreases importantly in lesional skin from AP patients.

HUVECs from newborns with a strong family history of myocardial infarction overexpress adhesion molecules and react abnormally to stimulating agents.

Atherosclerosis is a complex disease involved in major fatal events such as myocardial infarction and stroke. It is the result of interactions between metabolic, dietetic and environmental risk factors acting on a genetic background that could result in endothelial susceptibility. Our aim was to determine the patterns of expression of adhesion molecules and whether phosphatidylserine is translocated to the cell surface of human umbilical vein endothelial cells (HUVECs) isolated from healthy newborns born to parents with a strong family history of myocardial infarction under TNF-alpha or oxLDL stimulated conditions. Compared to control HUVECs, experimental cords showed: (a) a four-fold increase in VCAM-1 expression under basal conditions, which showed no change after stimulation with the pro-atherogenic factors; (b) a two-fold increase in basal P-selectin expression that reached a 10-fold increase with any of the pro-atherogenic factors; (c) a basal ICAM-1 expression similar to P-selectin that was not modified by the pro-atherogenic molecules; (d) a similar PECAM-1 expression. Unexpectedly, phospathidylserine expression in experimental cord HUVECs was significantly increased (211 817 versus 3354 TFU) but was not associated to apoptotic death as the percentage of dead cells induced by TNF-alpha treatment was very low (0.55 versus 9.87% in control HUVECs). The latter result was corroborated by TUNEL staining. T cell adherence to HUVECs was highly up-regulated in the genetically predisposed samples. The analysis of nonpooled HUVECs, from newborns to family predisposed myocardial-infarction individuals, might represent a useful strategy to identify phenotypical and functional alterations, and hopefully, to take early preventive actions.

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes.

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.

Enforced and prolonged CD40 ligand expression triggers autoantibody production in vivo.

CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39-kDa glycoprotein (CD154) expressed on the surface of activated T lymphocytes and is essential for thymus-dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40-CD40 ligand interaction in vitro and in vivo by using CD154-transfected L929 cells. In vitro assays showed that CD154-L929 cells can induce on B cells: IL-4-dependent proliferation, up-regulation of CD23, CD54 and class II molecules and can also rescue WEHI-231 B cell lymphoma from anti-IgM-induced apoptosis. Interestingly, in vivo assays revealed that when CD154-L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti-erythrocyte autoantibodies. Through B lymphocyte activation with CD154-transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40-CD154 in the induction of primary responses in vivo.

CD40-CD40 ligand interactions in vivo regulate migration of antigen-bearing dendritic cells from the skin to draining lymph nodes.

Whereas CD40-CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand -/- mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand -/- mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand -/- mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-alpha production and was corrected by injecting recombinant TNF-alpha or an agonistic anti-CD40 monoclonal antibody. Thus, CD40-CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-alpha production and play a vital role in the initiation of acquired T cell-mediated immunity.

Dendritic cells require T cells for functional maturation in vivo.

We examined dendritic cell (DC) status in SCID and RAG2 -/- mice to assess the influence of T cells on DC development and function in vivo. These mice have reduced numbers of DC in the epidermis and lymph nodes draining hapten-sensitized skin. Epidermal DC in these mice were defective in presenting antigen in vivo to adoptively transferred, hapten-sensitized T cells from normal mice. Likewise, draining lymph node DC were deficient in their capacity to stimulate naive T cells in vitro and in vivo. DC numbers as well as the impaired ability to present antigen in vivo, were corrected by reconstituting these animals with normal T lymphocytes, suggesting that T cells are crucial for normal DC maturation and function in vivo.

Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses.

We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor-alpha or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.

Entamoeba histolytica: liver invasion and abscess production by intraperitoneal inoculation of trophozoites in hamsters, Mesocricetus auratus.

Intraperitoneal inoculation of axenically cultured Entamoeba histolytica trophozoites constitutes an easy to perform, highly reproducible procedure for inducing amebic liver abscesses in hamsters. Efficiency in abscess production (95% of infected animals after 1 week) was similar to data reported using direct intrahepatic or intraportal inoculation. The morphological sequence of infection shows that amebas in the peritoneal cavity initially produce a large exudate constituted mainly of acute inflammatory cells. These cells form a rim of polymorphonuclear leukocytes surrounding the amebas, which adhere to the trophozoite and can sometimes be observed polarized to one end of the parasite, suggesting capping of surface receptors. Early stages are also characterized by the production of distant inflammatory reactions in the hepatic portal spaces. At 6 h postintraperitoneal inoculation, larger foci of inflammatory reactions surrounding amebas are developed in the peritoneum, extending to and damaging the liver surface membranes as well as the serosa of other internal organs. Thereafter, tissue damage progresses deeper into the liver parenchyma, and a few days later, coalescing granulomas and large necrotic areas are observed in the liver tissue. Based on the present morphological time-sequence study, we suggest that inflammatory cells associated with E. histolytica trophozoites play an important role in commencing the damage of liver sheaths and producing the subsequent parenchymal lesions. The simplicity and reliability of this model are important factors to consider when large numbers of experimentally induced amebic liver abscesses are needed.

Human interdigitating dendritic cells directly stimulate CD40-activated naive B cells.

Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40+ lineage-negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high-level expression of major histocompatibility complex class II, the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL-2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co-localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.

CD40 ligation counteracts Fas-induced apoptosis of human dendritic cells.

Dendritic cells (DC) are cells of the hematopoletic system specialized in capturing antigens and initiating T cell-mediated immune responses. We show here that human DC generated in vitro by culturing CD34+ cord blood progenitor cells in granulocyte macrophage colony stimulating factor plus tumor necrosis factor-alpha express the Fas antigen (APO-1, CD95) and undergo apoptosis upon triggering of Fas by mAb. However, only a proportion of the cells die in response to Fas ligation, an observation that may be related to the virtual absence of the bcl-2 protein in about half of the cells. Ligation of DC CD40 by culture on CD40L-transfected fibroblastic cells up-regulates the expression of bcl-2 and, concomitantly, renders DC virtually resistant to Fas-induced apoptosis. Parallel experiments with mature, interdigitating dendritic cells (IDC) isolated from tonsils revealed that IDC express Fas but do not enter into apoptosis following Fas ligation, a finding that may be explained by their high levels of bcl-2. Thus, upon encountering antigen-specific T cells, DC become resistant to Fas-induced apoptosis, as a consequence of CD40 ligation and possibly by mechanisms associated to the up-regulation of bcl-2 protein expression.

In vitro Entamoeba histolytica adhesion to human endothelium: a comparison using two strains of different virulence.

Extraintestinal dissemination of Entamoeba histolytica is frequently manifested by the life-threatening amebic liver abscess (ALA). The hepatic establishment of amebas implies invasion of blood vessels and contact with the endothelium. By means of a fluorescence-based quantitative adhesion assay, we assessed the binding to human endothelial cells of two E. histolytica strains of different virulence. The highly virulent strain (L-A) adhered substantially more strongly to unstimulated endothelium than the non-virulent one (BG3). Attachment of L-A was increased by treatment of endothelial cells with interleukin-1 beta (IL1 beta). Other proinflammatory cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha) did not modify the spontaneous adhesion capacity of amebas. For purposes of comparison we also performed adhesion of the parasites to skin fibroblasts. Adhesion to this cell type was quite low (< 10%). Parasite virulence, differential adhesive capacity to endothelial cells, and modulation of the latter phenomenon by proinflammatory factors (IL1 beta) may influence the evolution and outcome of extraintestinal amebiasis, especially hepatic abscesses.

CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells.

Human CD34+ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor-receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.