Substance P inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor

SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full-length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10?? M SP and ex vivo NK cells with 10?? M SP inhibited cytotoxic ability by ～20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down-modulated NCR expression nor affected triggering receptor-induced NF-κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor-induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.     

Thymic stromal lymphopoietin augments the cytolytic activity of the human natural killer cell line NK-92

Culturing the human natural killer cell line NK-92 for 24 h in the presence of thymic stromal lymphopoietin (TSLP) potentiated its cytotoxic capacity against the erythroleukemia cell line K562. Longer incubation times did not augment the NK activity any further. No synergistic effects with respect to either proliferation or cytotoxicity were observed when TSLP was mixed with suboptimal concentrations of IL-2. FACS analysis of the NK-92 cells indicated expression of TSLPR but not the other component of the TSLP receptor complex, namely IL-7Ralpha. Some of the surface molecules known to be involved in NK cell-mediated cytotoxicity were also monitored. None of the receptors analyzed altered their expression to any major extent upon culture in TSLP or IL-2. However, a limited number of NK-92 cells were observed that had a rather low CD94/NKG2A expression, which increased upon stimulation with TSLP or IL-2.     

Leucine rich repeats and calponin homology domain containing 1 inhibits NK-92 cell cytotoxicity through attenuating Src signaling

NK-92 cell line has been used as anti-tumor cytotoxic effector cells in immunotherapy. Leucine-rich repeats and calponin homology domain containing 1 (LRCH1) is a novel gene of which the function is unclear. In the present study, we investigated the role of LRCH1 in NK-92 cell cytotoxicity. LRCH1 was ablated in NK-92 cells through CRISP-Cas9-mediated knockout. LRCH1 knockout did not influence the basal behavior of NK-92 cells such as cell survival, expression of natural cytotoxicity receptors, and proliferation. However, upon the contact with tumor cells, LRCH1 knockout promoted NK-92 cell cytotoxicity to tumor cells. Besides, LRCH1 knockout increased the production of cytotoxic mediators such as IFN-γ, TNF-α, IL-2, and granzyme B in NK-92 cells after tumor cell contact. Similarly, LRCH1 knockout increased the production of cytokines and granzyme B upon NKp30 engagement. Further experiments revealed that LRCH1 knockout enhanced the activation of Src and Lck kinase which are important for natural killer cell cytotoxicity. The in vivo assay confirmed the up-regulation of the tumoricidal activity of LRCH1-/- NK-92 cells, as demonstrated by more robust tumor cell killing. Importantly, human primary natural killer cells exhibited a similar increase in the production of IFN-γ and TNF-α when LRCH1 was knocked out. In conclusion, our study revealed the role of LRCH1 as a negative regulator of NK-92 cell cytotoxicity.     

Interleukin-32 enhances cytotoxic effect of natural killer cells to cancer cells via activation of death receptor 3

Studies have demonstrated that the anti-tumour effect of natural killer (NK) cells is successful for patients with several cancers. Although interleukin-32 (IL-32) is endogenously expressed in NK cells, cytolytic function of NK cells against cancer cells has not been fully demonstrated. In the present study, we found that the growth of cancer cells was suppressed when colon cancer cells or prostate cancer cells were co-cultured with NK-92 cells, an NK cell line. We also found that the expression of tumour necrosis factor receptor 2 and death receptor 3 (DR3) was increased in PC3 cells, and the expression of FAS and DR3 was increased in SW620 cells by co-culture with NK-92 cells. However, cancer cell growth inhibition and IL-32 expression were abolished when cancer cells were co-cultured with NK cells transfected with small interfering (si) RNA of IL-32. DR3 expression was also diminished by co-culture with IL-32-specific siRNA-transfected NK-92 cells. Expression of APO3L, a ligand of DR3, was elevated in NK cells that were co-cultured with cancer cells. It was also found that expression of apoptosis-related proteins such as cleaved caspase-3 and bax was increased in cancer cells co-cultured with NK-92 cells, but their expression was abolished by co-culture with IL-32 siRNA-transfected NK-92 cells. Moreover, knockdown of DR3 in co-culture of NK-92 cells with cancer cells by siRNA or antibodies of DR3 and APO3L reversed the growth inhibitory effect of NK-92 cells. In conclusion, our study showed that IL-32 enhanced the cytotoxic effect of NK-92 cells on the cancer cells through activation of DR3 and caspase-3.     

Possible roles of KIR2DL4 expression on uNK cells in human pregnancy

PROBLEM: To investigate possible roles of the natural killer (NK) cell receptor killer immunoglobulin-like receptor (KIR)2DL4 expressed on uterine NK (uNK) cells during pregnancy, we investigated KIR2DL4 expression on uNK cells isolated from patients with early recurrent spontaneous abortion (RSA) and normal early pregnancy women, and functions of KIR2DL4 was analyzed in vitro. METHODS OF THE STUDY: Semi-quantitative RT-PCR analysis was introduced to detect KIR2DL4 messenger RNA (mRNA) expression on uNK cells. Cytotoxicity and cytokine production as the result of interaction of KIR2DL4 and its ligand human leukocyte antigen (HLA)-G were analyzed in vitro with lactic dehydrogenase releasing method and enzyme-linked immunosorbent assay, respectively. RESULTS: No significant difference in KIR2DL4 mRNA expression was observed, while the KIR2DL4 protein level in isolated uNK cells is much higher in normal controls than that in RSA patients. Data showed that HLA-G transfection could not reverse the lysis of uNK against HLA-G transfected K562 cells but induced cytokine production. Furthermore, we demonstrated that, via KIR2DL4, membrane-bound HLA-G could induce high cytotoxicity and cytokine production in a high cytotoxic, IL-2 dependent human NK cell line NK-92 cells. CONCLUSION: Our data suggest that KIR2DL4 might play a crucial implication for human pregnancy.     

乙型肝炎病毒C蛋白对NK细胞功能影响的体外研究

目的 研究乙型肝炎病毒C蛋白在NK细胞中的表达及其对NK细胞功能的影响.方法 用脂质体转染法将HBV C基因真核表达载体pHBI-CMV-HBC转入NK-92细胞,通过WesternBlot检测C基因在NK-92细胞中表达,用ELISA法检测NK细胞分泌IFN-γ水平,MTT法检测NK细胞对HepG2细胞的细胞毒作用.结果 Western Blot证实转染有pHBI-CMV-HBC的NK-92细胞能表达HBV C蛋白.转染了重组真核表达质粒的NK-92细胞IFN-γ水平均高于空质粒对照组和空白对照组(P<0.01).与两对照组相比,重组真核表达质粒转染组NK-92细胞对于HepG2细胞的细胞毒活性明显提高,差异具有统计学意义(P<0.01).结论 HBc基因瞬时高表达可影响NK-92细胞分泌IFN-γ和细胞毒功能.     

白血病细胞系NK细胞配体表达及其对NK-92细胞杀伤敏感性的研究

目的:检测白血病细胞系NK配体表达情况以及对NK-92细胞的敏感性,探讨NK配体表达与NK杀伤敏感性的关系。方法:通过半定量RT-PCR的方法,以β-actin为内参基因,分别检测U937、CEM、KG-1a、HL-60、NB4、Reh和LCL等7种细胞表面MICA、MICB、ULBP1、PVR、Nectin-2、LFA-3、LLT-1、HLA-E、HLA-F和HLA-G等10种NK配体的表达情况。通过流式细胞术,用CFSE和PI双染法,检测NK-92细胞对以上7种白血病细胞系的杀伤效应。结果:根据NK-92细胞对靶细胞的杀伤效应强弱,将以上7种白血病细胞系分成NK敏感组(U937,CEM,KG-1a)、中度敏感组(HL-60、NB4)和不敏感组(Reh,LcL)三组。NK配体PVR和HLA-F在组间的差异具有统计学意义(P值分别为0.017和0.016),不敏感组的PVR转录水平最低而HLA-F水平最高,其他配体间则无显著性差别。结论:本研究初步表明PVR和HLA-F是人为干预肿瘤细胞耐受NK细胞的两个靶点分子。     

Differential killing of pre-B acute lymphoblastic leukaemia cells by activated NK cells and the NK-92 ci cell line

The use of NK cells in adoptive therapy for malignant disease is an area of great potential. Currently the only NK cell line in clinical trials is NK-92, an activated NK cell line with a broad range of cytotoxicity against malignant cells. The activity of NK-92 against pre-B acute lymphoblastic leukaemias, however, is highly variable. In this study we compare the cytotoxic mechanisms and signalling pathways utilized by NK-92 ci and IL-2 activated NK cells to mediate killing of pre-B acute lymphoblastic leukaemia cell lines. Deficiencies in TNF family mediated apoptosis, phosphoinositide-3 kinase dependent and phosphoinositide-3 kinase independent killing limit the efficiency of NK-92 ci against pre-B acute lymphoblastic leukaemia cells. Importantly, treatment of the poorly killed leukaemia cells with TNF-alpha augmented both phosphoinositide-dependent and -independent cytolysis.     

CD226和9.1C3特异性抗体对NK-92细胞杀伤作用的影响

目的 :探讨NK细胞活化型受体CD2 2 6和NK细胞抑制型受体 9 1C3分子特异性抗体对活化NK细胞系NK 92杀伤的调节作用。方法 :通过间接免疫荧光染色和FCM分析 ,鉴定CD2 2 6和 9 1C3分子在NK 92细胞的表达 ,采用51 Cr释放实验和重导向杀伤实验观察CD2 2 6mAb和 9 1C3mAb对NK 92杀伤作用的影响。结果 :NK 92细胞为CD2 2 6阳性 ,9 1C3弱阳性。CD2 2 6mAb和 9 1C3mAb对NK 92自然杀伤作用没有明显的调节作用。在重导向杀伤实验中 ,CD2 2 6mAb能增强NK 92对P815细胞的杀伤作用 ,而 9 1C3mAb对NK 92的自然杀伤以及CD2 2 6mAb诱导的杀伤均没有抑制作用。结论 :NK细胞活化型受体CD2 2 6分子可能参与NK 92细胞杀伤作用的正向调节 ,而NK细胞抑制受体 9 1C3分子对NK 92细胞杀伤功能的影响不大。     

Phytoncides (wood essential oils) induce human natural killer cell activity

To explore the effect of forest bathing on the human immune system, we investigated the effect of phytoncides (wood essential oils) on natural killer (NK) activity and the expression of perforin, granzyme A and granulysin in human NK cells. We used NK-92MI cell, an interleukin-2 independent human NK cell line derived from the NK-92 cell, in the present study. NK-92MI cells express the CD56 surface marker, perforin, granzyme A, and granulysin by flow cytometry and are highly cytotoxic to K562 cells in chromium release assay. Phytoncides significantly increase cytolytic activity of NK-92MI cells in a dose-dependent manner and significantly increase the expression of perforin, granzyme A, and granulysin in the NK-92MI cells. Phytoncides also partially, but significantly, restore the decreased human NK activity and the decreased perforin, granzyme A, and granulysin expression in NK-92MI cells induced by dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide. Pretreatment with phytoncides partially prevents DDVP-induced inhibition of NK activity. Taken together, these data indicate that phytoncides significantly enhance human NK activity and this effect is at least partially mediated by induction of intracellular perforin, granzyme A, and granulysin.     

Phytoncides (Wood Essential Oils) Induce Human Natural Killer Cell Activity

To explore the effect of forest bathing on the human immune system, we investigated the effect of phytoncides (wood essential oils) on natural killer (NK) activity and the expression of perforin, granzyme A and granulysin in human NK cells. We used NK-92MI cell, an interleukin-2 independent human NK cell line derived from the NK-92 cell, in the present study. NK-92MI cells express the CD56 surface marker, perforin, granzyme A, and granulysin by flow cytometry and are highly cytotoxic to K562 cells in chromium release assay. Phytoncides significantly increase cytolytic activity of NK-92MI cells in a dose-dependent manner and significantly increase the expression of perforin, granzyme A, and granulysin in the NK-92MI cells. Phytoncides also partially, but significantly, restore the decreased human NK activity and the decreased perforin, granzyme A, and granulysin expression in NK-92MI cells induced by dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide. Pretreatment with phytoncides partially prevents DDVP-induced inhibition of NK activity. Taken together, these data indicate that phytoncides significantly enhance human NK activity and this effect is at least partially mediated by induction of intracellular perforin, granzyme A, and granulysin.     

Natural cytotoxicity in the neonate: high levels of lymphokine activated killer (LAK) activity.

In 28 healthy full-term newborns the percentage of circulating cells expressing the Leu7 antigen, the marker of natural killer (NK) cells, was significantly lower than in healthy adults. However, newborns and adults did not differ with regard to the percentage of cells reacting with the Leulla, Leullc and TEC NK-1, monoclonal antibodies directed against the IgG Fc receptor of killer cells. Spontaneous NK activity of neonatal cells was profoundly reduced compared to the adult. In contrast, antibody dependent cellular cytotoxicity and NK-like activity generated in mixed lymphocyte cultures were similar in the two groups and lymphokine-activated killer cell (LAK) activity was high in the neonate. Natural killing is thought to play an important role in antiviral immunity since the neonate has a deficient capacity to deal with viral infections. Consequently, the present data indicate either that spontaneous NK is the most informative in vitro measure of newborn natural cytotoxicity in vivo, or, alternatively, that natural killing is not as important in antiviral immunity as previously suggested.     

Mast cell deficient and neurokinin-1 receptor knockout mice are protected from stress-induced hair growth inhibition

Despite the lack of insight on distinct mediators in the skin orchestrating the pathophysiological response to stress, hair loss has often been reported to be caused by stress. Recently we revealed the existence of a brain-hair follicle axis by characterizing the neurokinin (NK) substance P (SP) as a central element in the stress-induced threat to the hair follicle, resulting in premature onset of catagen accompanied by mast cell activation in the skin. However, our understanding of possible SP–mast cell interactions in the skin in response to stress was limited since the receptor by which SP activates skin mast cells and the extent of mast cell mediated aggravation of SP remained to be elucidated. We now employed NK-1 receptor knockout mice (NK-1R^–/–) and mast cell deficient W/W^v mice and observed that stress-triggered premature induction of catagen and hair follicle apoptosis does not occur in NK1^–/– and W/W^v mice. Furthermore, the activation status of mast cells was less in stressed NK1^–/– mice than in wild-type control. Additionally, stress-induced upregulation of SP positive nerve fibers was absent in both NK-1R and W/W^v mice. These results indicate that the cross-talk between SP and mast cell activation via NK-1R appears to be the most important pathway in the regulation of hair follicle cycling upon stress response.     

TGF-β regulated leukemia cell susceptibility against NK targeting through the down-regulation of the CD48 expression

Transforming growth factor-β (TGF-β) is known to function as a dual role regulatory cytokine for being either a suppresser or promoter during tumor initiation and progression. In solid tumors, TGF-β secreted from tumor microenvironment acts as a suppresser against host immunity, like natural killer (NK) cells, to favor tumor evasion. However, besides solid tumors, the underlying mechanism of how TGF-β regulates leukemogenesis, tumor progression, immunoediting, and NK function is still not clear in detail. In this study, we found that TGF-β induced leukemia MEG-01 and U937 cells to become less sensitive to NK-92MI targeting by down-regulating CD48, a ligand for NK activating receptor 2B4, but not down-regulating other tumor-associated carbohydrate antigens (TACAs). In CD48-knockdown cells, cells responding to NK-92MI targeting displayed a phenotype of less NK susceptibility and cell conjugation. On the other hand, when NK cells were treated with TGF-β, TGF-β suppressed NK recognition, degranulation, and killing activity in time-dependent manner by regulating ICAM-1 binding capacity instead of affecting expressions of activating and inhibitory receptors. Taken together, both leukemia cells and immune NK cells could be regulated by TGF-β through suppressing leukemia cell surface CD48 to escape from host surveillance and down-regulating NK cell surface ICAM-1 binding activity to impair NK functions, respectively. Our results suggested that TGF-β had effect in leukemia similar to that observed in solid tumors but through different regulatory mechanism.     

Spatial association of prolyl oligopeptidase, inositol 1,4,5-triphosphate type 1 receptor, substance P and its neurokinin-1 receptor in the rat brain: an immunohistochemical colocalization study

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyzes proline-containing peptides shorter than 30 amino acids. It has been suggested that POP is associated with cognitive functions, possibly via the cleavage of neuropeptides such as substance P (SP). Recently, several studies have also linked POP to the inositol 1,4,5-triphosphate (IP(3)) signaling. However, the neuroanatomical interactions between these substances are not known. We used double-labeled immunofluorescence to determine the POP colocalization with SP, SP receptor (neurokinin-1 receptor, NK-1R) and IP(3) type 1 receptor (IP(3)R1) in the rat brain. Furthermore, since striatal and cortical GABAergic neurons are involved in SP neurotransmission, we studied the coexpression of POP, SP and GABA by triple-labeled immunofluorescence. POP was moderately present in IP(3)R1-containing cells in cortex; the colocalization was particularly high in the thalamus, hippocampal CA1 field and cerebellar Purkinje cells. Colocalization of POP with SP and NK1-receptor was infrequent throughout the brain, though some POP and SP coexpression was observed in cerebellar Purkinje cells. We also found that POP partially colocalized with SP-containing GABAergic neurons in striatum and cortex. Our findings support the view that POP is at least spatially associated with the IP(3)-signaling in the thalamus, hippocampus and cerebellar Purkinje cells. This might point to a role for POP in the regulation of long-term potentiation and/or depression. Moreover, the low degree of colocalization of POP, SP and its NK-1R suggests that a transport system is needed either for POP or SP to make hydrolysis possible and that POP may act both intra- and extracellularly.     

Colocalization of neuronal nitric oxide synthase and neurokinin-1 receptor in striatal interneurons in the rat

It has been established that nitric oxide synthase (NOS)-containing aspiny neurons constitute one class of interneurons in the striatum, and that substance P (SP)-containing projection neurons give off many axon collaterals within the striatum. In the present study, we investigated a morphological substrate of possible influences of SP-containing projection neurons upon NOS-containing interneuron in the rat striatum; colocalization of immunoreactivities for NOS and NK-1 type tachykinin receptor (NK1R: SP receptor) was examined by a double-immunofluorescence histochemistry. The vast majority (94.6%) of NOS-positive neurons showed NK1R immunoreactivity, whereas only smaller cells of NK1R-positive neurons (61.2% of NK1R-positive neurons) displayed NOS immunoreactivity. The results indicated that the NOS-containing interneurons were under direct control of SP-containing projection neurons in the striatum.     

Expression of the NK-1 receptor on islet cells and invading immune cells in the non-obese diabetic mouse

The underlying mechanistic causes of immune cell infiltration in the islets of Langerhans and beta cell failure in the non-obese diabetic (NOD) mouse is still to be completely revealed. Substance P (SP) is a substance known to have pro-inflammatory, endocrine, neuromodulatory and trophic effects, and its preferred receptor, the neurokinin receptor 1 (NK-1 R), is reported to be involved in extravasation of granulocytes and in inflammation and tissue derangement. Therefore, we have investigated the expression of NK-1 R during development of insulitis in the NOD mouse. We show that the magnitude of immunoreactivity scoring NK-1 R expression in the islets was increased in the 12-week-old NOD mouse. Expression of NK-1 R co-localized with expression of glucagon. In line with this expression pattern, we did not detect any effect of SP on glucose-induced insulin release. NK-1 R expression was particularly observed in islet cells in association with the clusters of immune cells. Expression of NK-1 R was also demonstrated in a fraction of the infiltrating B and T lymphocytes, as well as on infiltrating macrophages and dendritic cells. The observations show that the level of NK-1 R expression is increased in 12-week-old NOD mice, being correlated with the occurrence of islet mononuclear infiltration. Our data suggest that SP may act as a chemoattractant, contributing to the pathogenic mononuclear infiltration process in the NOD mouse. On the whole, the observations suggest that SP and the NK-1 R to certain extents are involved in the changes that occur during the development of insulitis in the NOD mouse.     

Priming effects of substance P on calcium changes evoked by interleukin-8 in human neutrophils

The neurokinin (NK) substance P (SP), which is a mediator of neurogenic inflammation, has been reported to prime human polymorphonuclear neutrophils (PMNs). The priming effects of SP on PMNs activated by recombinant interleukin-8 (rIL-8) were investigated. SP enhanced, in a dose- and time-dependent way, the rise in cytosolic free-calcium concentration, [Ca(2+)]i, evoked by the chemokine. The priming effects of SP were abolished by exposing PMNs to a calcium-free medium supplemented with EGTA. The C-terminal peptides SP(4-11) and SP(6-11) but not the N-terminal peptide SP(1-7) shared the priming effects of SP. The selective NK-1 receptor agonist [Sar-9, MetO2-11]SP mimicked the effects of SP, which were not reproduced by the selective NK-2 receptor agonist [betaAla-8]-NKA(4-10) or the selective NK-3 agonist senktide. Two selective NK-1 antagonists, CP96,345 and L703,606, dose dependently inhibited SP priming effects. These results demonstrated that SP primes PMNs exposed to rIL-8 and suggested that SP priming effects are receptor mediated.     

P物质及其受体的研究进展

神经肽P物质(SP)是速激肽家族的重要成员,通过与其受体相结合发挥生物学作用.P物质和其偏嗜性受体神经激肽受体-1(NK1R)广泛分布在中枢和外周神经系统及周围组织,SP通过与G蛋白耦联的受体相互作用调控多种功能.目前在mRNA水平和蛋白水平所发现的人NK1R只有两种:由5个外显子组成全长型受体(NKIR-FL)和C-...