躯体性应激和心理性应激对鼠脾NK细胞受体表达的影响

阐明躯体性应激和心理性应激降低NK细胞杀伤活性的机制及异同。应用Communication box系统分别使小鼠连续负荷躯体性应激和心理性应激后,以~(51)Cr释放法检测鼠脾NK、LAK细胞的杀伤活性;以流式细胞术检测鼠脾细胞、LAK细胞中NK细胞受体的表达水平。结果表明,躯体性应激和心理性应激均可降低鼠脾NK、LAK细胞的杀伤活性,但对脾细胞、LAK细胞中NK细胞受体表达的影响却不同,提示躯体性应激和心理性应激影响NK细胞功能的机制不同。     

LAK细胞

一、LAK细胞的概念 1980年Rosenberg等首先观察到荷有甲基胆蒽纤维肉瘤的C57BL/6小鼠脾细胞经含有IL-2的培液培养后,可获得较强的抗自身甲基胆蒽纤维肉瘤的细胞毒作用,而对自身正常细胞无杀伤作用,未经IL-2培养的脾细胞或NK细胞对纤维肉瘤无杀伤活性。1982年Grimm等首次提出将这种由IL-2激活的能够杀伤NK抵抗性肿瘤细胞的杀伤细胞称为淋巴因子激活的杀伤细胞(Lymphokine-activated killer cells)简称LAK细胞。脾脏细胞、淋巴结细胞、外周血淋巴细胞(PBL)均可经IL-2诱导出显著的     

D-半乳糖亚急性中毒拟衰老模型鼠免疫功能及生化指标变化的研究

目的研究D-半乳糖慢性中毒所致衰老模型鼠免疫功能与生化指标变化。方法制备D-半乳糖亚急性中毒导致的衰老鼠模型;给药第8周时以P815细胞腹腔注射免疫小鼠,诱导特异性CTL细胞并用MTT法检测CTL细胞杀伤活性;间隔1周后以10％淀粉腹腔注射,诱导腹腔巨噬细胞,吞噬中性红法检测腹腔巨噬细胞（Mφ）功能;给药第10周杀鼠,取胸腺计算胸腺指数;采用流式细胞术检测胸腺细胞凋亡情况;MTT法检测T淋巴细胞转化增殖功能与NK细胞杀伤活性;取脾细胞48h培养上清,检测IL-2、INF-γ、TNF三种TH1型细胞因子的活性;硫代巴比妥酸（TBA）法检测血清中丙二醛（MDA）含量,黄嘌呤氧化酶法检测血清中总超氧化物歧化酶（SOD）活性;ISP半自动生化分析仪检测血清中血糖（Glu）、总胆固醇（CHO）、甘油三酯（TG）的含量。结果衰老鼠胸腺指数低于对照组（P〈0．05）;胸腺细胞凋亡高于对照组（P〈0．05）;T淋巴细胞增殖率显著低于对照组（P〈0．05）;IL-2、INF-γ、TNF三种TH1型细胞因子的活性较对照组均显著降低（P〈0．05）;与对照组比较VE衰老鼠腹腔Mφ细胞吞噬功能明显降低（P〈0．05）、NK细胞杀伤活性也有所降低（P〈0．05）,但CTL细胞的杀伤活性变化不明显（P〉0．05）。衰老模型鼠血清中MDA含量显著高于正常对照组（P〈0．05）,衰老模型鼠血清总SOD含量显著低于正常对照组（P〈0．05）。模型鼠血清中的血糖、总胆固醇、甘油三酯含量显著高于正常对照组（P〈0．05）。结论D-半乳糖慢性中毒所致衰老模型鼠免疫功能明显减退,氧自由基MDA、SOD的改变及生化学指标血糖、血脂改变趋势与自然衰老的动物模型相一致。     

P物质在应激活化小鼠皮肤肥大细胞中的作用

目的探讨P物质(substance P,SP)在应激活化小鼠皮肤肥大细胞中的作用。方法应用Communication box系统使小鼠分别负荷足电击和心理性应激,采用甲苯胺蓝(toluidine blue)染色法、免疫组化ABC技术分别检测皮肤肥大细胞数及SP阳性神经纤维数,以ELISA法检测皮肤中SP含量,以I125放免法测定血浆类固醇浓度。结果躯体性应激和心理性应激均可使血浆类固醇升高,躯体性应激负荷鼠的血浆类固醇激素在第3d达高峰,而后逐渐下降;而心理性应激负荷鼠的血浆类固醇激素在第5d达高峰后逐渐下降。腹腔注射WIN51078可明显降低两种应激负荷所致的血浆类固醇浓度,且对足电击的抑制效果优于心理性应激。足电击和心理性应激均可有意义增强皮肤肥大细胞的活化,增加SP阳性神经纤维数。足电击可使皮肤SP含量呈现有意义的减少,而心理性应激则皮肤SP含量增加,这些变化可因非肽性NK1速激肽受体拮抗剂WIN51078的腹腔注射而抑制。结论SP参与了应激导致的肥大细胞活化机制,同时显示了速激肽受体拮抗剂可能会抑制应激导致的皮肤症状的恶化。     

重组腺病毒Ad-Mig基因治疗裸鼠肾细胞癌的实验研究

目的:研究腺病毒介导的小鼠Mig基因对BALB/c裸鼠肾细胞癌的抗肿瘤效果,探讨肾细胞癌治疗的新途径.方法:利用786-O肾癌细胞皮下注射BALB/c裸鼠建立肾细胞癌模型,应用携带Mig基因的重组腺病毒(Ad-Mig)直接进行瘤内注射治疗,观察裸鼠皮下肿瘤生长情况和荷瘤裸鼠的生存期;用乳酸脱氢酶(LDH)释放法检测CTL和NK的杀伤活性.结果:Mig基因能显著抑制荷瘤裸鼠皮下肿瘤的生长,并使鼠生存期明显延长,还能显著增强鼠脾细胞NK和CTL杀伤活性.结论:重组腺病毒Ad-Mig基因对鼠肾细胞癌有显著治疗效果.     

双歧杆菌脂磷壁酸对B16荷瘤小鼠NK细胞受体NKG2D及其配体的影响

目的探讨双歧杆菌脂磷壁酸（LTA）对黑色素瘤B16荷瘤小鼠NK细胞受体NKG2D及其配体的影响。方法将黑色素瘤B16细胞接种于C57BL／6小鼠皮下,待触及肿块后于荷瘤小鼠皮下注射双歧杆菌LTA。采用MTT、流式细胞术（FCM）、RT-PCR方法分别检测经双歧杆菌LTA处理后B16荷瘤小鼠NK细胞杀伤活性、NK细胞NKG2D受体蛋白表达以及肿瘤组织内Rae-1、H60 mRNA表达的变化。结果与对照组相比,经双歧杆菌LTA处理后,B16荷瘤小鼠的NK细胞杀伤活性增强（P〈0．05）,NK细胞受体NKG2D表达明显增加（P〈0．05）,肿瘤组织Rae-1、H60 mRNA表达上升（P〈0．05）,并具有浓度依赖性。结论双歧杆菌LTA能够增强B16荷瘤小鼠NK细胞的杀伤活性,其机制可能与上调NK细胞受体NKG2D的蛋白表达和肿瘤组织Rae-1、H60 mRNA的表达有关。     

双歧杆菌脂磷壁酸对化疗荷瘤小鼠免疫功能的影响

目的探讨双歧杆菌脂磷壁酸对5-氟尿嘧啶（5-FU）化疗肝癌H22荷瘤小鼠免疫的影响及其作用机制。方法双歧杆菌脂磷壁酸处理5-Fu化疗的H纶荷瘤Balb／c小鼠,MTT法检测NK细胞和CTL细胞杀伤活性;采用流式细胞仪检测荷瘤小鼠脾细胞中T亚群比例;用RT-PCR和Western Not方法分别检测荷瘤小鼠肿瘤组织Foxp3和TIM-3mRNA及蛋白的表达变化。结果荷瘤小鼠脾细胞中CD4^＋ CD25^＋Tmg比例高,并存在Foxp3和TIM-3 mRNA及蛋白的高表达,经双歧杆菌LTA和5．Fu处理后CD4^＋CD25^＋Tmg比例下降,Foxp3和TIM-3mRNA及蛋白表达水平也均呈下调趋势,但5-FU单独处理后的荷瘤小鼠脾细胞中CD4^＋细胞比例也减少,NK细胞和CTL杀伤率均降低,而双歧杆菌LTA处理后CD4^＋细胞比例,NK细胞和CTL杀伤率却明显增加。二者联合处理也能增加CD^＋细胞比例及NK细胞和CTL杀伤率。结论双歧杆菌脂磷壁酸联合5-FU可通过增强NK细胞和CTL杀伤能力,同时抑制TIM-3／TIM-3L途径,降低CD4^＋CD25^＋Tmg的免疫抑制活性,增强机体细胞免疫来提高化疗抗肿瘤效果,减轻化疗副作用,增强宿主对化疗的耐受性,从而提高抗肿瘤作用。     

松子壳多糖对小鼠主要免疫细胞功能的影响

目的探讨松子壳多糖（pine nut shell polysaccharide,PSP）对小鼠主要免疫细胞的影响。方法应用MTT法测定PSP对小鼠脾淋巴细胞的毒性和对ConA或LPS诱生小鼠脾T、B淋巴细胞的转化,用中性红吞噬试验测定腹腔巨噬细胞的吞噬功能,应用乳酸脱氢酶释放法测定NK细胞的杀伤活性。结果 PSP对脾细胞毒性很低,各种浓度对小鼠脾淋巴细胞的增殖均有较强的促进作用（P〈0.01）;PSP在浓度50～300μg/mL时,明显促进T淋巴细胞的转化（P〈0.01）,但是当浓度达到300μg/mL时表现出一定的抑制作用（P〉0.05）;PSP在25～200μg/mL时显著促进了小鼠脾B淋巴细胞的转化（P〈0.05）,但是当浓度达到300μg/mL时,抑制作用极显著（P〈0.01）;不同浓度均可以增强小鼠腹腔巨噬细胞吞噬中性红的能力及其代谢功能,当浓度在100μg/mL时能显著的促进巨噬细胞吞噬中性红的能力（P〈0.01）;PSP在浓度100～300μg/mL时,能极显著的促进NK细胞对Yac-1的杀伤作用（P〈0.01）,当浓度达到400μg/mL,对NK细胞杀伤性的促进作用开始减弱。结论 PSP对小鼠脾淋巴细胞的毒性较低,能增强免疫细胞活性,有望成为新一代免疫调节剂。     

失重条件对小鼠NK细胞体外生物活性的影响

建立在失重环境下NK细胞的培养体系,探讨失重培养条件对NK细胞活性的影响。取C57BL/6小鼠脾,分离NK细胞,在正常重力和失重状态下旋转细胞两种培养体系中培养NK细胞48 h。以Yac-1细胞为靶细胞,采用MTT法检测其杀伤活性;半定量PCR分析穿孔素、颗粒酶的转录水平。与正常重力培养对照相比,失重培养的NK细胞杀伤活性显著降低,其穿孔素和颗粒酶B的转录水平均显著低于正常重力培养组,穿孔素/GAPDH结果为失重培养组0.625±0.042;正常重力培养组1.054±0.036(P<0.01);得到颗粒酶B/GAPDH结果为失重培养组0.700±0.042;正常重力培养组1.068±0.058(P<0.01)。成功建立了NK细胞体外培养体系,在失重环境下NK细胞杀伤活性降低。     

手术应激与免疫抑制

手术前后时期的心理应激和生理应激均可抑制细胞免疫功能,主要表现为T淋巴细胞和自然杀伤（natural killer,NK）细胞的数量减少和活性减弱。手术创伤越大,对免疫的抑制作用越强,因而对手术后的痊愈有明显影响。一般而言,手术应激所致的免疫抑制可以恢复,恢复的时间主要取决干手术创伤的大小。目前认为,手术引起免疫抑制的机制主要与下丘脑-垂体-肾上腺皮质轴、交感神经系统、细胞因子、阿片肽和T细胞信号分子有关。选用某些不具有免疫抑制作用的止痛药物,以及蛋白酶抑制剂,可以防治手术应激所致的免疫抑制作用。     

Effector and precursor phenotypes of lymphokine-activated killer cells in mice with severe combined immunodeficiency (scid) and athymic (nude) mice

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.     

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44^lo) or elevated (CD44^hi) expression of CD44 are generated and that the CD44^hi cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.     

The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from NK cell- and lymphokine-activated killer cell-mediated cytolysis

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K(b)-expressing B78H1 targets also reduced LAK cell activity, while H-2D(b) offered no protection. Importantly, blocking with F(ab')(2) specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2(b) SCID and B6 mice established that NK1.1(+)TCR(-) NK and NK1.1(+)TCR(+) LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1(+) NK/T cells.     

Similarities and distinctions between murine natural killer cells and lymphokine-activated killer cells

Pretreatment of mice with rabbit anti-asialo GM1 removes both natural killer (NK) effector cells and NK cells responsive to interleukin 2 (IL-2). Spleen cells from these mice do possess normal lymphokine-activated killer (LAK) activity. Young mice (less than 3 weeks of age) do not have NK activity and do not possess IL-2-inducible NK effector cells. Similarly to anti-asialo GM1-treated mice, LAK cells can be generated from these mice. While these experiments indicate clear distinctions between a certain level of NK and LAK precursors, the distinctions are not as clear when analyzing mice congenitally deficient in NK cells. Beige mice which lack NK effector cells and IL-2-inducible NK cells also lack the ability to generate LAK cells. The relationships and differences between NK- and LAK-cell precursors and effectors are discussed.     

Inverse relationship in H-2-associated lysis between NK cells and rIL-2-activated killer cells: evidence from in vitro killing and metastatic experiments

We investigated the manner in which rIL-2 induced effectors in vitro (LAK cells), which, like NK cells, lyse targets nonspecifically and discriminate nonself, and how H-2 as the self-marker affects the LAK cell killing mechanism. NK cells showed an appreciably higher killing activity to B16 melanoma H-2- cells than to H-2+ cells. In contrast, LAK cells lysed more efficiently to H-2+ cells. The in vivo experiments showed that the NK cells prevented pulmonary metastasis of B16 H-2- cells in the normal syngeneic host, whereas the transferred LAK cells had a preferential inhibitory effect on the pulmonary metastasis of B16 H-2+ cells in the immunodeficient syngeneic hosts. Taken together, these results show that the H-2-encoded or H-2-associated molecules contribute to the triggering signal in the lysis by LAK cells, whereas the NK cells recognize the reduced self H-2 expression on the targets, thereby contributing to a trigger of the lysis.     

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia

Summary The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.     

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.     

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100–120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1–15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5×10⁴ Cetus units twice daily for 4–5 days), 5-FU augmented (up to 37-fold, days 1–9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3–4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3–9), as determined by limiting-dilution cultures with IL-2 (for 7–8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6–9 days after treatment with 5-FU, Adriamycin or dacarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6–12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1-LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.