Sperm with damaged membranes are profoundly at risk to have caspases 8, 9 and 3 activated

PROBLEM:  It is almost dogma that IL-2 is not expressed at the M–F interface during normal pregnancy. However, recent results by ours and others clearly showed that IL-15-Th1 type cytokine which shares many similarities with IL-2 is expressed at the interface. IL-15 can affect cytolytic activity of maternal decidual lymphocytes which heavily infiltrate maternal decidua during the first trimester pregnancy. These cells are in a direct contact with trophoblastic cells. IL-18 is a recently discovered Th1 type cytokine with many interesting functions. The aim is to examine IL-18 distribution at the interface and its potential in up-regulating peripheral blood (PB) and decidual lymphocytes (DL) cytotoxicity. Th1 activated lymphocytes are LAK cells and they can kill by both Perforin and Fas pathways in non MHC restricted manner.
METHODS:  PBL and DL were obtained from elective pregnancy termination of pregnancy. IL-18 and IL-18R expression was detected by flow cytometry and immunohistology and cytolytic potential by cytotoxicity against K-562 (NK sensitive) and P815 (NK resistant) cell lines.
RESULTS:  IL-18 positive cells were found in the suspension of Decidual adherent cells and IL-18 R expressions at the trophoblastic cells of villi. Both IL-15 and IL-18 are increasing cytolytic potential of PBL and DL against NK sensitive cell line. Decidual lymphocytes are activated cells with the potential of killing NK resistant but LAK sensitive lines and this is mediated by both perforin and Fas pathways.
CONCLUSIONS:  Physiological and pathophysio- logical role(s) of cytolytic pathways and Th1 cytokines (IL-15 and IL-18) at the interface will be discussed.     

Progesterone directly and indirectly affects perforin expression in cytolytic cells

PROBLEM: Decidual lymphocytes (DL) expressing the cytolytic molecule perforin represent approximately 55% of DL in the first trimester of human pregnancy. Progesterone dominates this phase of pregnancy and controls the production of uterine cytokines and growth factors. The aim of this study was to investigate the role of progesterone and progesterone-induced blocking factor (PIBF) on perforin expression in DL and peripheral blood lymphocytes (PBL). METHOD OF STUDY: Perforin expression was analyzed in PBL and DL incubated either in culture medium or with decidual adherent cells (DAC) and peripheral blood adherent cells (PBAC) and their supernatants with or without progesterone or PIBF. Perforin was detected by flow cytometry in PB and in decidual first trimester pregnancy lymphocytes. RESULTS: Progesterone in high concentrations directly affects perforin expression in DL but not in PBL. Progesterone in a concentration dependent manner indirectly blocks perforin expression in DL and PBL cultured with adherent cells or their supernatants. PIBF blocked upregulation of perforin expression of DL cultured with DAC, but none of those cultured with PBAC. Similarly, PIBF was inefficient when PBL or DL were cultured with PBAC. CONCLUSION: Progesterone present in a high concentration locally at the maternal-fetal interface modulates perforin expression in the first trimester pregnancy DL.     

Progesterone induced blocking factor (PIBF) mediates progesterone induced suppression of decidual lymphocyte cytotoxicity

PROBLEM: Progesterone induced blocking factor (PIBF) is a mediator of progesterone that blocks peripheral blood lytic natural killer (NK) activity. Progesterone or PIBF stimulated decidual macrophages block up-regulation of perforin expression in decidual lymphocytes (DL). Therefore, we investigated whether progesterone regulates cytotoxicity of DL. METHOD OD STUDY: Decidual mononuclear cells were cultured with progesterone. PIBF, progesterone and anti-PIBF antibody or in the medium only. Cytolytic activity of non-adherent DL was measured by PKH-26 (red) 2 hr cytolytic assay and flow cytometry. Perforin positive DL were detected by immunofluorescency and PIBF-positive cells by immunohistology. RESULTS: Progesterone and PIBF, in a dose-dependent manner decreased cytotoxicity of DL against K-562 targets, and perforin egzocytosys was blocked. Anti-PIBF antibodies reversed the progesterone mediated reduction in cytolytic activity of DL. PIBF positive cells were found in first trimester pregnancy decidua. CONCLUSION: The results indicate possible role for PIBF, as a mediator of progesterone in regulation of DL cytolytic activity at the maternal-foetal (M-F) interface.     

Decidual macrophages are the population of decidual adherent cells which regulates perforin expression in cytolytic cells.

PROBLEM: We have shown that addition of decidual adherent cells (DAC) to the culture of decidual lymphocytes (DL) prevents the downregulation of perforin expression in these cells. Because DAC are a mixture of various cell populations, the aim is to analyze immunophenotypic characteristics of DAC and to determine which cell population is involved in the regulation of perforin expression. METHOD OF STUDY: First trimester pregnancy decidual cells were obtained by enzymatic tissue digestion. Decidual cells and peripheral blood lymphocytes (PBL) were centrifuged on Ficoll-Hypaque density gradient and cultured overnight to obtain adherent cells, which were analyzed by flow cytometry and immunocytochemically. RESULTS: Almost all peripheral blood adherent cells (PBAC) (ca 90%) expressed monocyte/macrophage markers but only 10-20% of DAC. The rest of DAC expressed markers of stromal cells. HLA-DR depleted population of DAC (stromal cells only) could not prevent downregulation of perforin expression in cultured DL and PBL. CONCLUSION: Decidual macrophages are involved in the regulation of perforin expression in DL.     

Modulation of perforin expression in the decidual and peripheral blood cytotoxic lymphocytes in culture.

PROBLEM: Perforin (P) is a cytolytic molecule located in intracellular granules of cytotoxic lymphocytes both in the peripheral blood and decidua of pregnancy. The aim was to analyze the kinetics of P expression during in vitro culture and modulation of P expression by adherent cells, their supernatants and mitogen (PHA) stimulation. METHOD OF STUDY: P (intracellular antigen) was detected by flow cytometry in the suspension of first trimester pregnancy peripheral blood lymphocytes (PBL) and decidual lymphocytes (DL). RESULTS: A decrease of the percentage of P+ cells was obtained after 1 hr incubation and was prevented by addition of 30% of decidual adherent cells (DAC) or their supernatants. Upregulation of P expression was obtained when, in addition to adherent cells, DL and PBL were stimulated by PHA. DAC present in the culture in physiological concentrations prevent downregulation of P expression. CONCLUSION: DAC located in the vicinity of decidual cytotoxic lymphocytes, owing to their unique ability to produce a wide range of substances on demand, contribute to the high level of P expression in the decidua of pregnancy.     

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNgamma production by NK cells. mRNA coding for IFNgamma is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNgamma protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNgamma.     

复发性自然流产患者绒毛及蜕膜组织中白细胞介素8的表达

目的:研究复发性自然流产患者绒毛及蜕膜组织中白细胞介素8(interleukin-8,IL-8)的表达。方法:应用免疫组化法及图像分析技术,对30例复发性自然流产患者绒毛、蜕膜组织中IL-8的蛋白表达进行定位及半定量分析;H- E染色后光镜下观察绒毛及蜕膜组织的形态学变化。结果:在绒毛组织中,IL-8蛋白定位于绒毛上皮细胞的细胞质内,且病例组的表达明显高于对照组;在蜕膜组织中,病例组蜕膜细胞的胞质内可见IL-8蛋白表达,且高于对照组;H- E染色可见病例组绒毛组织的滋养层变薄,细胞变性甚至坏死、嗜酸性增强,绒毛中轴纤维化程度增强;蜕膜组织中蜕膜细胞失去细胞间连接,部分蜕膜细胞解体、核消失。结论:病例组绒毛及蜕膜组织中IL-8蛋白表达的增强与复发性自然流产有关,IL-8可能参与复发性自然流产的病理过程。     

Progesterone-induced blocking factor inhibits degranulation of natural killer cells.

PROBLEM: During the first trimester of pregnancy, nonclassical (CD3-, CD56+, CD16-, perforin [P]bright+) natural killer (NK) cells comprise the major decidual lymphocyte population. These cells, in spite of their high perforin content, exert a low cytolytic activity. Peripheral blood lymphocytes of healthy pregnant women produce progesterone-induced blocking factor (PIBF), which inhibits NK activity. PIBF-producing cells are likely to be present in decidua and might contribute to low decidual NK activity. METHOD OF STUDY: Decidual cells obtained from elective pregnancy termination were double labeled for CD56 and PIBF. We tested the effect of PIBF on perforin liberation by activated peripheral blood NK cells. RESULTS: Sixty percent of decidual lymphocytes were CD56 + and expressed PIBF at the same time. PIBF-treated and untreated peripheral blood NK cells were incubated with K-562 cells, and perforin content of target conjugated NK cells was detected with immunocytochemistry. PIBF treatment of peripheral blood lymphocytes significantly reduced lysis of K-562 cells. Among target bound lymphocytes in PIBF-treated samples, we found a significantly (P < 0.01) higher rate of P+ cells than in untreated samples. CONCLUSIONS: These data suggest that PIBF inhibits cytotoxicity of NK cells via a block of degranulation, and since decidual NK cells are PIBF+, it cannot be ruled out that this effect of PIBF contributes to low decidual NK activity.     

Decidual natural killer cell tuning by autologous dendritic cells

PROBLEM: Dendritic cells (DC)/natural killer (NK) cells interactions in the deciduas of early human pregnancies were analyzed in vitro. METHOD OF STUDY: Phenotype, cytokine expression and/or cytolytic mediators' expression were measured by flow cytometry in NK and DC from the freshly isolated decidual mononuclear cells or after their purification and co-culture in vitro. Proliferation of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled CD56(+) cells was analyzed by flow cytometry after the co-culture with CD1a(+) or CD83(+) DC. RESULTS: Decidual CD1a(+) cells show less mature phenotype with no expression of CD197, lower expression of CD80 and CD86 and higher expression of CD206 and CD195 in comparison to CD83(+) cells. Interleukin (IL)-15, interferon-gamma and tumor necrosis factor-alpha productions were higher in immature than mature DC, whereas IL-10 and IL-18 were equally produced in both subpopulations. Immature DC increase perforin, FasL and TRAIL protein expression and proliferation of NK cells, but decrease their intracellular IL-15 production. Mature DC caused less efficient proliferation of NK cells, and did not affect cytokine and cytolytic mediator expression. CONCLUSION: These results suggest that decidual CD1a(+) cells regulate and shape NK cell function more profoundly than CD83(+) cells in decidua.     

KIR downregulation by IL‐12/15/18 unleashes human NK cells from KIR/HLA‐I inhibition and enhances killing of tumor cells

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin‐like receptor (KIR)‐mediated self‐inhibition of human NK cells by HLA‐I–expressing tumor cells. Here, we show that stimulation of NK cells with IL‐12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re‐induced already 3 days after re‐culture in IL‐2. Reduced KIR2DL2/L3 expression on IL‐12/15/18–activated NK cells resulted in less inhibition upon antibody‐mediated KIR engagement and increased CD16‐dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL‐12/15/18–stimulated NK cells against tumor cells expressing cognate HLA‐I molecules. NK cells pre‐activated with IL‐12/15/18 were previously shown to exert potent anti‐tumor activity and memory‐like long‐lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL‐12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self–HLA‐I due to decreased KIR expression, highlighting the potency of IL‐12/15/18–activated NK cells for anti‐tumor immunotherapy protocols.     

弓形虫感染对人早孕母胎界面细胞因子转录水平的影响

研究弓形虫感染时IL-4、IL-10在绒毛滋养层细胞和IFN-γ在蜕膜NK细胞中mRNA水平的变化,从而探讨弓形虫感染致不良妊娠的分子免疫机制。选取正常妊娠5~10周妇女行人工流产术后的绒毛及蜕膜组织14例,利用绒毛组织分离滋养层细胞和蜕膜组织分离蜕膜NK细胞。分别将绒毛滋养层细胞和蜕膜NK细胞平均分为实验组和对照组,加入弓形虫培养和单独培养48 h后,采用实时荧光定量聚合酶链式反应(real-time PCR)检测滋养层细胞IL-4、IL-10 mRNA表达水平和蜕膜NK细胞IFN-γmRNA表达水平。结果:实验组和对照组IL-4、IL-10、IFN-γmRNA平均表达水平分别为(0.05±0.02)、(0.06±0.03)、(0.33±0.09)和(0.31±0.13)、(0.18±0.06)、(0.08±0.03),三者之间均具有显著性差异(P<0.05、P<0.01、P<0.05);实验组IFN-γ/IL-10、IFN-γ/IL-4的比值分别为(3.96±0.84)和(3.21±0.41),对照组则分别为(0.60±0.21)、(0.78±0.25),两组间同样均具有显著性差异(P<0.01、P<0.01)。早孕期弓形虫感染可致母胎界面Th1型细胞因子表达增高,Th2型细胞因子表达降低,从而打破正常妊娠状态下二者的平衡,可能是早孕期弓形虫感染导致不良妊娠的重要分子机制。     

IL‐18, but not IL‐15, contributes to the IL‐12‐dependent induction of NK‐cell effector functions by Leishmania infantum in vivo

Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK‐cell response in mice that required TLR9‐positive myeloid DC and IL‐12, but no IFN‐α/β. Here, we investigated whether IL‐15 or IL‐18 mediate the activity of IL‐12 or function as independent activators of NK cells. In contrast to earlier studies that described IL‐15 as crucial for NK‐cell priming in response to TLR ligands, the expression of IFN‐γ, FasL, perforin and granzyme B by NK cells in L. infantum‐infected mice was completely preserved in the absence of IL‐15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN‐γ secretion, cytotoxicity and FasL expression of NK cells from infected IL‐18^?/? mice were significantly reduced compared with controls, but, unlike IL‐12, IL‐18 was not essential for NK‐cell effector functions. Part of the NK‐cell‐stimulatory effect of IL‐12 was dependent on IL‐18. We conclude that IL‐15 is not functioning as a universal NK‐cell priming signal and that IL‐18 contributes to the NK‐cell response in visceral leishmaniasis. The cytokine requirements for NK‐cell activation appear to differ contingent upon the infectious pathogen.     

Perforin-Expressing Lymphocytes in Peripheral Blood and Decidua of Human First-Trimester Pathological Pregnancies

PROBLEM: We have shown previously that the decidua of first-trimester human pregnancy is heavily infiltrated with perforin-positive cells. The aim was to detect expression of perforin in both decidual lymphocytes (DL) and peripheral blood lymphocytes (PBL) in the first trimester of pathological pregnancies: Anembryonic pregnancy and missed abortion. METHOD: Decidual tissue from a normal pregnancy group and from pathological pregnancies was obtained by vaginal curettage. Perforin (an intracellular antigen) and the cell surface antigens CD3, CD4, CD8, CD16, CD56, CD11c, and CD45RA were quantified simultaneously by flow cytometric analysis. RESULTS: In the missed abortion group, we found: 1) a relative decrease in the frequency of both CD4⁺P⁺ cells and CD56⁺P⁺ cells as well as the mean fluorescence intensity for perforin; 2) a relative increase of CD16⁺P⁺ PBL cells; and 3) a relative increase of CD4⁺ cells in PBL compared with anembryonic pregnancy and normal pregnancy. There was also a significant relative decrease in the proportion of CD4⁺ and CD8⁺ cells among perforin-positive PBL in both anembryonic pregnancy and missed abortion. CONCLUSION: Our results show that significant decreases in the prevalence of perforin-positive lymphoid cells, their subpopulations, and mean fluorescence intensity for perforin are associated with pregnancy failure.     

Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity

Interleukin 12 (IL-12)-activated NK1.1+TCRalpha beta+ (NKT2) and NK1.1+TCRalpha beta- (NK) cells exhibit cytotoxic activity against a wide variety of tumor cells in the absence of prior sensitization. Here we demonstrate that the integrin adhesion receptor LFA-1 (CD11a/CD18) regulates the cytotoxic activity of IL-12-activated NKT and NK cells against YAC-1 and EL-4 tumor cells. Differentiation in vivo and the expression of the cytolytic effector molecules perforin and Fas-L were comparable in both IL-12-activated NKT and NK cells from LFA-1-/ - and LFA-1+/+ mice. However, LFA-1-/-IL-12-activated NKT and NK cells showed impaired conjugate formation with target cells. These results provide the first genetic evidence for a role for an adhesion receptor in killing by IL-12-activated NK cells.     

In vivo expression of perforin by natural killer cells during a viral infection. Studies on uveitis produced by herpes simplex virus type I.

A potent cytolytic pore-forming protein (PFP, perforin, or cytolysin) is associated with the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The role of PFP/perforin in cytolytic reactions carried out in vivo is still unclear. Here, the authors performed immunohistochemical analysis using antibodies monospecific for perforin and made use of a murine uveitis model produced by intracameral inoculation of herpes simplex virus I (HSV-I). The main cell infiltrate found in the anterior segment of virus-inoculated eyes consisted of Thy-1+/asialo GM1+/CD8-/CD4- cells, presumably representing NK cells. Perforin staining was detected mainly in cells bearing this phenotype. Perforin was only detected in cells displaying the large granular lymphocyte morphology. A small number of perforin-positive cells (less than 5%) colabeled as CD8+, indicating that these cells could have belonged to the CTL lineage. These observations show for the first time the presence of perforin-containing NK cells in tissues of animals undergoing acute viral infections.     

Leukotriene B4 augments interleukin-2 receptor-beta (IL-2R beta) expression and IL-2R beta-mediated cytotoxic response in human peripheral blood lymphocytes.

Interleukin-2 can induce cytolytic activity in human peripheral blood lymphocytes and this activation is mediated by the beta chain of the interleukin-2 receptor-beta (IL-2R beta). Leukotriene B4 (LTB4) is a potent lipid inflammatory mediator which induces IL-2 and interferon-gamma (IFN-gamma) production from T cells. We examined the ability of LTB4 to modulate IL-2-induced cytolytic activity. Peripheral blood lymphocytes which had been preincubated for 24 hr in the presence of LTB4 responded to 100-fold lower concentrations of IL-2 with an augmentation of natural killer (NK) cell cytotoxic activity. Furthermore, incubation of lymphocytes with graded concentrations of LTB4 augmented the proportion of IL-2R beta+ cells. Peak activity was seen at 10 nM LTB4 and was comparable to that of PHA. By two-colour cytofluorometry, the increased expression of IL-2R beta was found predominantly on CD56+ cells and to a lesser extent on CD8+ cells, while CD4+ cells were unaffected. These observations were correlated at the messenger RNA (mRNA) level with increased IL-2R beta mRNA accumulation following stimulation of purified CD56+ and CD8+ cells with LTB4. CD56-, CD8- cells did not respond to LTB4 by increased IL-2R beta mRNA accumulation. Our data indicate, for the first time, that LTB4 can markedly increase the sensitivity of non-major histocompatibility complex (MHC)-restricted cytotoxic lymphocytes to IL-2, in terms of IL-2-dependent cytotoxic responses, and that this sensitivity is associated with augmented IL-2R beta gene message and cell surface expression.     

Interleukin-11 signaling is required for the differentiation of natural killer cells at the maternal-fetal interface.

Interleukin-11 (IL-11) is a multifunctional hematopoietic growth factor that has been implicated in the control of reproduction. Studies on IL-11 receptor-alpha (IL-11R alpha)-deficient mice showed that female mice are infertile due to defective decidualization. In this report, we evaluated the development of decidual cells, immune cells, and the vasculature associated with the implantation site of IL-11R alpha-deficient mice; with the aim of better understanding the nature of the fertility defect. Messenger RNAs for decidual differentiation, such as decidual prolactin-related protein and prolactin-like protein-J are expressed in the IL-11R alpha mutant. However, the number of decidual cells expressing these genes is decreased in the mutant compared with the wild-type control. Although, trophoblast cells differentiate and express placental lactogen-I in the IL-11R alpha-deficient uterine environment, they fail to progress and expand in number. Defects in the organization of the decidual vasculature were also apparent in the IL-11R alpha mutant uterus. The most dramatic effect of IL-11 signaling was on the hematopoietic environment of the uterine decidua. Differentiated/perforin-expressing uterine natural killer (NK) cells were virtually absent from implantation sites of IL-11R alpha mutant mice. NK cell precursors were capable of homing to the IL-11R alpha-deficient uterus and a known regulator of NK cell differentiation; IL-15 was expressed in the IL-11R alpha mutant uterus. Splenic NK cells from IL-11R alpha mutant mice were also able to respond to IL-15 in vitro. Thus, the defect in NK precursor cell maturation was not intrinsic to the NK precursor cells but was dependent upon the tissue environment. In summary, IL-11 signaling is required for decidual-specific maturation of NK cells.     

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.