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Kerstin H Mair Andrea Müllebner Sabine E Essler J Catharina Duvigneau Anne K Storset Armin Saalmüller Wilhelm Gerner 《Veterinary research》2013,44(1):13
Natural Killer (NK) cells play a crucial role in the early phase of immune responses against various pathogens. In swine so far only little information about this lymphocyte population exists. Phenotypical analyses with newly developed monoclonal antibodies (mAbs) against porcine NKp46 recently revealed that in blood NKp46- and NKp46+ cells with NK phenotype exist with comparable cytotoxic properties. In spleen a third NKp46-defined population with NK phenotype was observed that was characterised by a low to negative CD8α and increased NKp46 expression. In the current study it is shown that this NKp46high phenotype was correlated with an increased expression of CD16 and CD27 compared to the CD8α+NKp46- and NKp46+ NK-cell subsets in spleen and blood. Additionally NKp46high NK cells expressed elevated levels of the chemokine receptor CXCR3 on mRNA level. Functional analyses revealed that splenic NKp46high NK cells produced much higher levels of Interferon-γ and Tumor Necrosis Factor-α upon stimulation with cytokines or phorbol-12-myristate-13-acetate/Ionomycin compared to the other two subsets. Furthermore, cross-linking of NKp46 by NKp46-specific mAbs led to a superior CD107a expression in the NKp46high NK cells, thus indicating a higher cytolytic capacity of this subset. Therefore porcine splenic NKp46high NK cells represent a highly activated subset of NK cells and may play a profound role in the immune surveillance of this organ. 相似文献
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Jamila Elhmouzi-Younes Anne K. Storset Preben Boysen Fabrice Laurent Fran?oise Drouet 《Veterinary research》2009,40(6)
Natural killer (NK) cells are key components of the innate immune system with their killing and cytokine producing abilities. Bovine NK cells have been characterized as NKp46+/CD3− lymphocytes, but little is known about these cells in neonatal calves. As the newborn calf, with an insufficiently developed acquired immunity, has to employ the innate immune system, we wanted to investigate whether neonate NK cells had the same characteristics as cells from older calves. Freshly isolated neonate and calf NK cells presented the same resting CD2+/CD25low/CD8−/low phenotype. Neonates less than 8 days old had one third of the circulating NKp46+ cells of older calves, but the NK cells proliferated more actively in vitro in the presence of interleukin (IL)-2 or IL-15. Moreover, neonate NK cells were more cytotoxic both in an NKp46 mediated redirected lysis assay and in direct killing of a bovine cell line MDBK when cultured in the presence of IL-15. Neonate and calf NK cells cultured in the presence of IL-2 and then stimulated with IL-12 produced similar dose-dependent interferon (IFN)-γ amounts, while IL-15 cultured NK cells did not give such a response whatever the age. However, neonatal NK cells cultured in IL-15 and stimulated by IL-12 concomitantly with cross-linking of NKp46, produced 4 to 5 times more IFN-γ than calf NK cells. These data suggest that although present in lower number at birth, neonate NK cells are fully functional and are more responsive to IL-15 and activation through the NKp46 receptor than NK cells from older calves. 相似文献
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Noronha LE Harman RM Wagner B Antczak DF 《Veterinary immunology and immunopathology》2012,147(1-2):60-68
The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population. 相似文献
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Natural killer (NK) cells have received much attention due to their cytotoxic abilities, often with a focus on their implications for cancer and transplantation. But despite their name, NK cells are also potent producers of cytokines like interferon-gamma. Recent discoveries of their interplay with dendritic cells and T-cells have shown that NK cells participate significantly in the onset and shaping of adaptive cellular immune responses, and increasingly these cells have become associated with protection from viral, bacterial and parasitic infections. Furthermore, they are substantially present in the placenta, apparently participating in the establishment of normal pregnancy. Consequently, NK cells have entered arenas of particular relevance in veterinary immunology. Limited data still exist on these cells in domestic animal species, much due to the lack of specific markers. However, bovine NK cells can be identified as NKp46 (CD335) expressing, CD3(−) lymphocytes. Recent studies have indicated a role for NK cells in important infectious diseases of cattle, and identified important bovine NK receptor families, including multiple KIRs and a single Ly49. In this review we will briefly summarize the current understanding of general NK cell biology, and then present the knowledge obtained thus far in the bovine species. 相似文献
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Yi-Chun Lin Yi-Chun Huang Yu-Shan Wang Rong-Huay Juang Kuang-Wen Liao Rea-Min Chu 《Veterinary immunology and immunopathology》2010,133(2-4):144-153
Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8+ peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8+ cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3+, CD4?, CD21?, CD5lo, α/βTCR+, and γ/δTCR? contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8? cells. This subset of CD8+ lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8? cells. In contrast, CD8+ cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8+ cells are an important subset associated with NK cytotoxic activity. 相似文献
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Jared R. Patch Pervaiz A. Dar Ryan Waters Felix N. Toka Jose Barrera Christopher Schutta Ganesh Kondabattula William T. Golde 《Comparative immunology, microbiology and infectious diseases》2014
Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46+ cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3+ cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV. 相似文献
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M Campos C R Rossi H Bielefeldt Ohmann T Beskorwayne N Rapin L A Babiuk 《Veterinary immunology and immunopathology》1992,32(3-4):205-223
Interleukin-2 (IL-2) treatment of cells and generation of non-major histocompatibility complex (MHC)-restricted cytotoxic cells from peripheral blood mononuclear leukocytes (PBML) was studied. Effector-target conjugate assays demonstrated that bovine PBML bound but did not lyse K562, HL60S and HL60R cells unless activated with IL-2. The magnitude of IL-2-activated killing of tumor cells as well as the magnitude of antibody-dependent cellular cytotoxicity depended on the IL-2 concentration. A short treatment (12-18 h) of effector cells with IL-2 was sufficient for development of cytotoxic activity. Withdrawal of IL-2 from the culture resulted in a reduction of cytotoxic activity that could be restored by further addition of IL-2. Cytotoxic activity of IL-2-activated populations obtained after nylon wool or Sephadex G-10 passage, and Percoll gradient centrifugation of PBML suggests that lymphokine-activated killer (LAK) cell activity in PBML is mainly mediated by a non-adherent lymphocyte lacking markers for B-cells. Positive and negative selection experiments using cell sorting confirmed these findings and demonstrated that the cell responsible for LAK cell activity in cattle belongs to a non-monocyte, non-B, CD2+ lymphocyte population. Furthermore, cytotoxic activity could not be generated in CD2+ populations enriched for cells expressing molecules equivalent to human and murine CD4 and CD8. These findings suggest that effector cells mediating non MHC-restricted cytotoxicity in cattle prevail in a population bearing a CD2+, CD4-, CD8- phenotype and that this population depends on the continuous presence of IL-2 for optimal cytotoxic function. 相似文献
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Helen T. Michael Daisuke Ito Valarie McCullar Bin Zhang Jeffrey S. Miller Jaime F. Modiano 《Veterinary immunology and immunopathology》2013
NK cells are non-T, non-B lymphocytes that kill target cells without previous activation. The immunophenotype and function of these cells in humans and mice are well defined, but canine NK cells remain incompletely characterized. Our objectives were to isolate and culture canine peripheral blood NK cells, and to define their immunophenotype and killing capability. PBMC were obtained from healthy dogs and T cells were depleted by immunomagnetic separation. The residual cells were cultured in media supplemented with IL-2, IL-15 or both, or with mouse embryonic liver (EL) feeder cells. Non-T, non-B lymphocytes survived and expanded in these cultures. IL-2 was necessary and sufficient for survival; the addition of IL-15 was necessary for expansion, but IL-15 alone did not support survival. Culture with EL cells and IL-2 also fostered survival and expansion. The non-T, non-B lymphocytes uniformly expressed CD45, MHC I, and showed significant cytotoxic activity against CTAC targets. Expression of MHC II, CD11/18 was restricted to subsets of these cells. The data show that cells meeting the criteria for NK cells in other species, i.e., non-T, non-B lymphocytes with cytotoxic activity, can be expanded from canine PBMC by T-cell depletion and culture with cytokines or feeder cells. 相似文献
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Line Olsen Preben Boysen Caroline Piercey ?kesson Gjermund Gunnes Timothy Connelley Anne K Storset Arild Espenes 《Veterinary research》2013,44(1):109
Natural killer (NK) cells are important for immune protection of the gut mucosa. Previous studies have shown that under pathologic conditions NK cells, T cells and dendritic cells are found co-localised in secondary lymphoid organs where their interaction coordinates immune responses. However, in the gut-associated lymphoid tissues (GALTs), there are few detailed reports on the distribution of NK cells. Sheep harbour several types of organised lymphoid tissues in the gut that have different functions. The ileal Peyer’s patch (IPP) functions as a primary lymphoid tissue for B cell generation, while the jejunal Peyer’s patches (JPPs) and colon patches (CPs) are considered secondary lymphoid tissues. In the present study, we analysed tissues from healthy lambs by flow cytometry and in situ multicolour immunofluorescence, using recently described NCR1 antibodies to identify ovine NK cells. Most NCR1+ cells isolated from all tissues were negative for the pan T cell marker CD3, and thus comply with the general definition of NK cells. The majority of NCR1+ cells in blood as well as secondary lymphoid organs expressed CD16, but in the GALT around half of the NCR1+ cells were negative for CD16. A semi-quantitative morphometric study on tissue sections was used to compare the density of NK cells in four compartments of the IPPs, JPP and CPs. NCR1+ cells were found in all gut segments. Statistical analysis revealed significant differences between compartments of the primary lymphoid organ IPP and the secondary lymphoid organs of the JPPs and CP. NK cells co-localised and made close contact with T cells, dendritic cells and other NK cells, but did not show signs of proliferation. We conclude that NK cells are present in all investigated segments of the sheep gut, but that presence of other innate lymphoid cells expressing NCR1 cannot be excluded. 相似文献
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Ovine haemopoietic progenitor cells that form colonies (CFC) in soft agar cultures were compared to more mature bone marrow cells for their level of expression of the adhesion receptor molecules ovine (ov) CD44, ov CD11a (LFA-1) and ov CD58 (LFA-3) as well as the 175-antigen using specific monoclonal antibodies. Ov CD44, ov CD11a and ov CD58 were expressed on all CFC of the myeloid (non-erythroid) series, whereas ov CD44 and ov CD11a expression was very low or absent from a small number of blast and erythroid series CFC. Within the mature non-erythroid population of myeloid cells, neutrophils retained a low level of expression of ov CD11a. Most CFC representing all lineages strongly expressed the ov CD44 antigen. In contrast, the majority of CFC lacked the 175-antigen, as did bone marrow lymphocytes, basophils and mast cells. This property of CFC was exploited in a negative selection technique using panning and immunomagnetic beads to select CFC from other bone marrow cells with a 116-125-fold enrichment, 12-14% purity and 29-40% yield. These results demonstrate that ovine CFC express some of the molecules necessary to allow adhesion to haemapoietic stromal cells and vascular endothelium in the tissues. Future studies will concentrate on the function of the adhesion receptor molecules in medullary and extra-medullary haemopoiesis and inflammatory cell development in sheep. 相似文献
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Kampen AH Olsen I Tollersrud T Storset AK Lund A 《Veterinary immunology and immunopathology》2006,113(1-2):53-63
Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ gammadelta TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor alpha chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of gammadelta T cells from birth to approximately 5 months of age. However, the absolute numbers of gammadelta T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of gammadelta T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute gammadelta T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life. 相似文献
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Gunnes G Valheim M Press CM Tverdal A Storset A 《Veterinary immunology and immunopathology》2003,94(3-4):177-183
Two approaches to the quantitative analysis of cell population markers in tissues are flow cytometry and image morphometry. To compare these methods, sheep lymph nodes were collected and analysed for CD8+ and CD21+ cell populations, which were selected to represent dispersed and concentrated cell populations, respectively. These two populations were measured as a percentage of total cell count (flow) or total tissue area (morphometry). The two populations were also measured as a percentage of respective base populations (CD2+ cells for CD8 and MHC II+ cells for CD21). A simple linear regression analysis showed that when the cell populations were assessed as a percentage of total cell count or total area, measurements obtained with flow and morphometry only correlated significantly with the dispersed CD8+ population and not with the highly concentrated CD21+ population. However, when the cell populations were assessed as a percentage of their base population, measurements obtained with flow and morphometry showed a significant correlation for both the dispersed and concentrated cell populations. This study demonstrates that measurements of lymph node cell populations obtained with the two methods are comparable, but that tissue distribution of cell populations should be considered, when the unit of measurement is chosen. 相似文献
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A L Berg A Johannisson M Johansson A Hein M Berg R D?rries 《Veterinary immunology and immunopathology》1999,68(2-4):241-253
Borna disease virus (BDV) is a neurotropic agent with capacity to cause encephalomyelitis in a wide range of animal species, including horses and cats. Recent studies also point to a link between BDV and human neuropsychiatric disorders. The pathogenesis of Borna disease (BD) has been proposed to be immune-mediated, mainly through the effects of cytotoxic T cells. We used flow cytometric analysis in order to characterize the peripheral and intracerebral T cell immune response in cats naturally infected with BDV. Our results show the presence of two different CD8+ cell populations (CD8+low and CD8+high) in the blood, spleen and brain of these cats. In the brain, CD8+low cells predominated over CD8+high cells. Since CD8+low cells have been suggested to represent a non-MHC-restricted T cell population, the recruitment of such cells to the brains of BDV-infected cats could possibly be of importance for the clearance of virus from neurones. 相似文献
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Sedgmen BJ Lofthouse SA Scheerlinck JP Meeusen EN 《Veterinary immunology and immunopathology》2002,86(3-4):215-220
Immunohistological characterisation of ovine rectal tissue has revealed the presence of lymphoid follicles, predominantly in the submucosa, that closely resemble those found in intestinal Peyer's patches (PPs). Distinct T (CD4+, CD8+, gammadelta-TCR+) and B (CD21+, CD45R+) lymphocyte staining patterns were observed within and around follicles of the rectal mucosa. In addition, IgA+ and IgE+ cells were also found at this tissue site, with both phenotypes commonly residing in the lamina propria. RT-PCR examination of the cytokines expressed in the rectal mucosal tissue revealed consistently high levels of TGFbeta and IL-8 mRNA, low levels of IL-2 mRNA and no detectable IL-4 mRNA. The presence of lymphoid follicles, IgA+ plasma cells and IgA-inducing cytokines in rectal tissue of sheep indicate that this may be a suitable route for delivering mucosal vaccines. 相似文献
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Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3−) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis. 相似文献
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Ferret-Bernard S Lacroix-Lamandé S Remot A Metton C Bernardet N Charley B Drouet F Laurent F 《Veterinary research》2011,42(1):19
ABSTRACT: At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40. 相似文献