Duration of intralipid's suppressive effect on NK cell's functional activity

BACKGROUND: In vitro investigations have revealed the ability of intralipids to suppress natural killer (NK) cytotoxicity. Evidence from both animal and human studies suggests that intralipid administered intravenously may enhance implantation and maintenance of pregnancy when the patient has an abnormal NK cell level or function. PROBLEM: The aim of this study was to establish the duration and efficacy of Intralipids suppressive effect on NK cell functional activity. METHOD OF STUDY: Fifty patients with abnormal NK activity results (NKa) received intralipid 20% i.v. (9 mg/mL total blood volume -corresponds to 2 mL of intralipid 20% diluted in 250 mL saline; or 18 mg/mL - corresponds to 4 mL of intralipid 20% diluted in 250 mL saline) infusions and their NKa were tested periodically. The determination of NK cell function was performed by flow cytometry using K562 cells as targets. RESULTS: Fifty women with abnormal NKa-testing received intralipid infusions. 39 (78%) showed NKa suppression within the normal range the first week after infusion, 11 (22%), showed suppression, but still above the normal threshold. They received second infusion 2-3 weeks later. In 10, the Nka activity was normalized the following week. Four patients had three intralipid infusions in 2-week periods in between and after the third infusion, and all showed NKa normal activity. In 47 patients the suppressive effect of the Intralipid after the normalization of NKa lasted between 6 and 9 weeks, in two patients this benefit lasted 5 weeks, and in one patient the effect was 4 weeks. CONCLUSION: Intralipid is effective in suppressing in vivo abnormal NK-cell functional activity. The results suggest that Intralipid can be used successfully as a therapeutic option to modulate abnormal NK activity in women with reproductive failure.     

Elevated Peripheral Blood Natural Killer Cells Are Effectively Downregulated by Immunoglobulin G Infusion in Women With Recurrent Spontaneous Abortions

PROBLEM: We investigated the hypothesis that elevated peripheral blood natural killer cells (NK) are decreased by immunoglobulin G infusion (IVIg) therapy in women with recurrent spontaneous abortions (RSA) and elevated NK cells. METHODS: Seventy-three women with RSA and elevated NK cells received IVIg therapy (400 mg/Kg/day for 3 days ever 4 wks) and anticoagulation treatment. Peripheral blood immunophenotype assay by flow cytometry was done prospectively prior to and 7 days after first IVIg therapy, every 2 wks until 20 wks gestation and then monthly. Controls were 95 women with RSA and normal NK cells who received anticoagulation treatment. RESULTS: (1) 86.3% of women with elevated NK cells who received the IVIg and anticoagulation therapy had a successful pregnancy outcome; (2) Peripheral blood CD56+ NK cells and CD56+/16+ NK cells were significantly suppressed 7 days post IVIg infusion (P < 0.0005); (3) Pre-IVIg infusion levels of other lymphocyte subsets were not different as compared with those of 7 days post-IVIg therapy; (4) Women who delivered a liveborn infant with IVIg therapy demonstrated downregulation of peripheral blood NK cells (CD56+, CD56+/16+) during early pregnancy when compared to women who miscarried the index pregnancy (P < 0.05); (5) Women with normal NK cells who miscarried while on anticoagulation therapy demonstrated significantly elevated CD56+ NK cells during early pregnancy as compared with that of women who delivered a liveborn infant (P < 0.05); (6) CD19+ B cells were significantly downregulated during pregnancy in women with anticoagulation and IVIg therapy when compared to women with anticoagulation therapy (P < 0.05). CONCLUSION: Downregulation of NK cells in women with RSA is associated with a favorable pregnancy outcome. Peripheral blood NK cells (CD56+, CD56+/16+) are effectively suppressed after IVIg therapy. Women with RSA and high NK cells benefit from IVIg therapy and experience suppression of CD56+ and CD56+/16+ NK cells.     

Intravenous Immunoglobulin Inhibits Natural Killer Cell Activity In Vivo in Women With Recurrent Spontaneous Abortion

We previously reported elevation of natural killer (NK) cells in women with recurrent spontaneous abortion (RSA) of immune etiology. In this study, we investigated the effect of intravenous immunoglobulin G (IVIg) on peripheral blood NK activity in vivo in women with RSA. Blood was drawn prior to and 7–11 days after IVIg therapy in eight women with RSA. NK activity was measured using K562 as target cells for ⁵¹Cr-release assays. Serum IgG concentrations were also measured. All received 400 mg/kg/day of IVIg for 3 consecutive days. 1) Seven of eight women became pregnant. Five delivered a live born infant. Three out of five women (60%) who delivered a live born infant showed a significant inhibition of NK cytotoxicity post IVIg and the rest did not show any changes; 2) NK cytotoxicity was significantly increased in a woman who miscarried again; 3) A woman who miscarried a chromosomally abnormal fetus showed a significant inhibition of NK cytotoxicity after IVIg; and 4) Serum IgG concentration increased significantly from 9.3 ± 3.0 mg/ml to 23.5 ± 5.1 mg/ml post IVIg therapy. IVIg effectively inhibits peripheral blood NK activity in vivo. These results are consistent with our previous finding showing that IVIg inhibits NK cell activity in vitro. Women with RSA and elevated NK cells may benefit from IVIg treatment.     

Natural killer cell function of human neonatal lymphocytes.

Human natural killer cell (NK cell) activity against K-562 target cell line was evaluated in full term cord blood (n = 30) and adult peripheral blood (n = 20) using 51Cr release assay. The level of NK cell activity was lower in cord blood compared to adult controls (39.6 +/- 11.4% vs 27.4 +/- 11.8% at effector:target ratio 50:1). Adult males showed a significantly higher NK activity compared to females. No sex difference was observed in cord blood. Furthermore, partially purified human leucocyte interferon (IFN alpha) increased in vitro NK cell function of both adult and newborn lymphocytes. The present results indicate that the appearance and maturation of human NK cells occurs during the intrauterine life of the human fetus.     

ORIGINAL ARTICLE: Suppression of Natural Killer Cell Cytotoxicity in Postpartum Women

Citation Groer M, El‐Badri N, Djeu J, Harrington M, Van Eepoel J. Suppression of natural killer cell cytotoxicity in postpartum women. Am J Reprod Immunol 2010; 63: 209–213 Problem Natural Killer (NK) cell numbers and cytotoxicity are suppressed during pregnancy. Little is known about postpartum NK number and function. Method of study Postpartum women (n = 39) were studied at one week and then monthly over the first six postpartum months. The standard natural killer cell cytotoxicity assay (NKCA) was performed. This is a Cr51 release assay from K562 cells cultured with peripheral blood mononuclear cells (PBMCs). Results Data indicate suppression of NK cytotoxicity in postpartum women. Cytotoxicity at each effector:target (E:T) ratio showed a drop from 1 week postpartum, reaching a nadir at around 2 months, and a trend towards recovery of cytotoxicity from 3 to 6 months. Lytic units (LUs) from pre‐incubated cells from postpartum women were lower than age‐matched, non‐pregnant, non‐postpartum controls through the fifth postpartum month. Conclusion These data suggest that the postpartum period, like pregnancy, is characterized by decreased NK cytotoxicity activity. This suppressed NK cytotoxic effect may result as a response to interaction with tolerized fetal microchimeric cells accumulated during pregnancy in maternal blood and tissues.     

Depressed natural killer cell function in thermally injured adults: successful in vivo and in vitro immunomodulation and the role of endotoxin.

Natural killer (NK) cells mediate host defense against infections and are regulated by interleukin 2 (IL-2) and other factors. We studied NK cell function in burn patients using a 51Cr release assay with K562 target cells. We found that peripheral blood lymphocytes from burn patients had depressed NK activity (target cell lysis = 22.0 +/- 3.1% vs 39.8 +/- 3.2% in healthy volunteers, P less than 0.001) and also a lower response to IL-2 (28.9 +/- 3.8% vs 53.2 +/- 4.3%, P less than 0.001). Thirteen burn patients were randomly assigned to receive either standard therapy or 5 days of intravenous polymyxin B in addition to standard therapy. After 2 weeks, the patients not receiving polymyxin B had a significant decline in peripheral blood NK activity (P less than 0.01) and response to IL-2 (P less than 0.05), while no decline in NK cell activity was seen in patients who received polymyxin B. Sera from burn patients was found to suppress the NK activity of lymphocytes from healthy adults by 5-75%. After using affinity chromatography to remove endotoxin, the sera from burn patients no longer suppressed NK cell activity. Circulating endotoxin appears to be involved in the suppression of NK activity in burn patients.     

Preconception peripheral natural killer cell activity as a predictor of pregnancy outcome in patients with unexplained infertility

PROBLEM: Preconception high peripheral natural killer (NK) cell activity in women with recurrent spontaneous abortion can predict subsequent miscarriages. We have examined prospectively, for the first time, the pregnancy rate in patients with unexplained infertility by measuring the peripheral NK activity. METHOD OF STUDY: We tested the peripheral NK activity of 94 infertile women who despite treatment were unable to conceive for 6 or more months (mean; 2.4 years). Peripheral NK activity was measured by a chromium-51 release cytotoxicity assay. Women were followed for 2 years and assessed. RESULTS: In 77 patients who were followed for 2 years, 28 had conceived but 49 did not. The peripheral NK activity of the group that became pregnant (mean +/- S.D.; 34.5 +/- 13.8%) was significantly lower than that of non-conception group (42.3 +/- 13.3%, P = 0.017). CONCLUSIONS: Our finding suggests that elevated peripheral NK activity in patients with unexplained infertility is a risk factor for attaining pregnancy success.     

G-CSF-mobilized CD34+ cells cultured in interleukin-2 and stem cell factor generate a phenotypically novel monocyte

To study the early stages of development from stem cells of the CD56+ cell population [which includes natural killer (NK) cells], granulocyte-colony stimulating factor-mobilized peripheral blood CD34+ cells from healthy donors were sorted to >99% purity and cultured in the presence of stem cell factor and interleukin (IL)-2. After 3 weeks in culture, the majority of cells acquired CD33, with or without human leukocyte antigen-DR and CD14. In 20 stem cell donors tested, 8.7 +/- 8.8% of cells were CD56+. Two major CD56+ subsets were identified: CD56(bright), mainly CD33- cells (7+/-10%, n=11) with large, granular lymphocyte morphology, and CD56dim, mainly CD33+ (2.5+/-2, n=11) cells with macrophage morphology. The CD56bright population had cytoplasmic granzyme A but lacked killer inhibitory receptor, suggesting they were immature NK cells. The CD56dim, CD33+, population lacked NK markers. They may represent a minor subset of normal monocytes at a developmental stage comparable with the rare CD56+ CD33+ hybrid myeloid/NK cell leukemia. Consistent with a monocyte nature, CD56dimCD33+ proliferated and produced a variety of cytokines upon lipopolysaccharide stimulation, including IL-8, IL-6, monocyte chemoattractant protein-1, and macrophage-derived chemokine but not interferon-gamma. In a short-term cytotoxicity assay, they failed to kill but powerfully inhibited the proliferation of the NK-resistant cell line P815. The generation of CD56+ cells was negatively regulated by hyaluronic acid and IL-4, indicating that extracellular matrix may play an important role in the commitment of CD34+ cells into CD56 myeloid and lymphoid lineages.     

Defective killer cell activity in patients with chronic active Epstein-Barr virus infection

Natural killer (NK) cell activity, lymphokine activated killer (LAK) activity and Epstein-Barr virus specific cytotoxic T lymphocyte (EBV-CTL) activity were examined in 10 children with chronic active EB-virus infection and an adult with persistently positive early antigen-antibody to EB-virus. NK cell activity against erythroleukemia cell line K-562 was significantly (p less than 0.005) lower in the patients (22.3 +/- 8.5%, mean +/- SD) than in normal controls (40.4 +/- 15.9%). Spontaneous cytotoxicity against an EB-virus transformed autologous lymphoblastoid cell line was 15.0 +/- 7.6% in the patients, and was comparable to spontaneous cytotoxicity activity in normal controls (11.7 +/- 4.3%). LAK activity against Raji cells was significantly (p less than 0.02) lower in the patients (14.6 +/- 11.4%) than in normal controls (29.2 +/- 15.9%). EBV-CTL activity against an EB-virus transformed autologous lymphoblastoid cell line was significantly (p less than 0.005) lower in the patients (11.8 +/- 5.5%) than in seropositive normal controls (33.7 +/- 14.7%). No regression of the lymphoblastoid cell line was observed when EBV-CTL activity of the patients was tested by regression assay. It is conceivable that defects in both EB-virus specific and nonspecific killer cell activities play important roles in the pathogenetic abnormalities which allow EB-virus infection to progress to a chronic active state.     

Detection of soluble HLA-G molecules in plasma and amniotic fluid

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.     

Massive Intravenous Immunoglobulin Treatment in Women with Four or More Recurrent Spontaneous Abortions of Unexplained Etiology: Down-Regulation of NK Cell Activity and Subsets

PROBLEM: The aims of this study were to investigate the efficacy of massive intravenous immunoglobulin (MIVIg) treatment for women with recurrent spontaneous abortion (RSA) of unexplained etiology, and to investigate changes in peripheral natural killer (NK) cell activity and subsets. METHOD OF STUDY: MIVIg treatment was performed in 18 pregnancies from 15 women with 4 or more consecutive RSA of unexplained etiology. NK cell activity and subsets were assessed in 8 of the pregnancies. RESULTS: 14 pregnancies resulted in live births and 4 resulted in abortions with chromosome abnormality. The pre-infusion NK cell activity (mean + SD. 40.9 + 17.0%) at 4.4 +/- 0.5 weeks of gestation (GW) decreased to 15.0 +/- 7.90% at post-infusion status (5.4 +/- 0.5 GW). Pre-infusion percentages of CD56+ CD16- cells (3.5 +/- 2.1%) and CD56+ CD16- cells (16.8 +/- 8.8%) decreased to 3.0 +/- 2.2% and 11.1 +/- 6.9%, respectively, after MIVIg treatment. CONCLUSIONS: MIVIg treatment was effective in all 14 pregnancies from RSA women of unexplained etiology, excluding 4 abortions with chromosome abnormality. Peripheral NK cell activity and subsets were suppressed by MIVIg treatment.     

A central role for phosphoinositide hydrolysis in activating the lytic mechanism of human natural killer cells

Using neomycin, which inhibits phosphoinositide breakdown, cytotoxicity mediated by natural killer (NK) cells was suppressed in a dose-dependent manner, with complete inhibition at 16 mM. Generation of inositol phosphates in effector cells after target cell binding was inhibited in the presence of neomycin. The formation of effector to target cell conjugates was not affected. Neomycin-induced inhibition of NK killing was abolished when TPA was added to the cytotoxic assays. This reconstitution was dependent upon extracellular Ca2+. When the intracellular free Ca2+ level in effector cells was reduced from 73 +/- 11 nM to 43 +/- 3 nM (n = 4) using the Ca2+ indicator dye, Quin 2, NK killing was markedly reduced. Inhibiting the enzyme diacylglycerol (DG) kinase in effector cells with 10 microM R59022 (DG kinase inhibitor) potentiates NK killing, suggesting an increase in protein kinase C (PKC) activity due to accumulation of DG. The PKC inhibitor, H-7, suppressed NK killing in a concentration-dependent manner. These results demonstrate that phosphoinositide metabolism is an early event and its derived second messengers play a central role in activating the lytic mechanism of NK cells.     

Direct visualisation and quantification of cellular cytotoxicity using two colour flourescence.

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, was used to label various target cell lines and propidium iodide (PI) uptake was used to assay cell death. Natural killer (NK), lymphokine activated killer (LAK) as well as targeted T cell mediated cellular cytotoxicity were quantified using the fluorescence method and compared to results obtained with the 51chromium (51Cr) release assay. A good correlation was found after an assay period of 4-8 h indicating that the fluorescence method is a reliable alternative to the 51Cr release assay.     

Interleukin-1 and interleukin-2 production in resistant and susceptible inbred mice infected with Trypanosoma congolense.

Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in cell-mediated cytotoxicity. Spontaneous cytotoxicity in a 2-hr assay against K562 target cells was shown to be largely mediated by contaminating natural killer (NK) cells. Treatment of adherent cells with NK-specific monoclonal antibody anti-Leu-11 plus complement abolished almost completely the spontaneous cytotoxicity. Spontaneous cytotoxicity by adherent cells was also reduced when the phorbol ester PMA was present in the assay. On the other hand, PMA induced a cytotoxic response in NK-cell depleted adherent cells after prolonged 18 hr incubation. The cell population responsible for this dichotomous effect of PMA on adherent cell-mediated cytotoxicity was shown to be monocytes, as revealed by monoclonal antibody treatment. Pure NK cell preparations were not affected by PMA in their cytolytic capacities. Reactive oxygen species are not involved in NK-cell mediated cytotoxicity, while PMA stimulated the monocytes to exert cytolysis and suppressed NK cells by the generation of these highly toxic oxygen products. Hydrogen peroxide especially seemed to be the mediator in this oxygen-dependent monocyte-mediated cytotoxicity and NK-cell suppression.     

Modulation of human natural killer cytotoxicity by influenza virus and its subunit protein.

The influence of intact influenza virus and purified detergent solubilized haemagglutinin (HA) subunits from these viruses on human natural killer (NK) cell activity was examined. Effector cells incubated with whole influenza virus for 18 hr initiated the production of alpha interferon which was associated with the enhancement of NK cell activity. In contrast, purified influenza virus HA suppressed NK activity in a dose-dependent manner, when added at the onset of the cytotoxicity assay, or when used to pre-treated effector cells prior to assay for cytotoxicity against K562 target cells. Effector cells exposed to influenza HA for 90 min, washed and re-incubated in fresh medium for up to 18 hr, failed to regain their cytotoxicity. Suppression of NK cell cytotoxicity could not be ascribed to direct toxicity of HA preparations or residual detergent and preservative in these preparations. The augmented cytotoxicity of activated human effector cells was also susceptible to suppression by virus HA, and pretreatment of human PBL effector cells with HA for 90 min, prior to exposure to human alpha interferon caused NK effector cells to become refractive to the enhancing effects of HIFN. That direct interaction between influenza virus HA and effector cells was a requirement for suppression of activity was shown in experiments using Bromelain-released influenza HA, which would not be expected to bind to cells and which failed to suppress NK cell activity.     

Natural cytotoxicity in systemic lupus erythematosus: mechanisms of suppression by inhibitory serum factors.

Spontaneous cytotoxicity mediated by natural killer (NK) cells is impaired in several human diseases including systemic lupus erythematosus (SLE). The present study was designed to describe factors in SLE sera which suppress the NK function of unfractionated mononuclear cells and NK enriched suspensions. NK activity was determined in 19 SLE patients and 25 normal controls by a standard chromium release assay. Sera obtained from SLE patients suppressed normal NK activity by an average of 29.4%. The presence of anti-lymphocyte antibodies (ALA) of the IgM class which were reactive with unfractionated mononuclear cells or the NK cell enriched OKM1 positive subset correlated with serum-mediated suppression. NK inhibitory SLE sera did not interfere with normal effector-target conjugate formation. These results demonstrate the modulatory effects of immune aggregates and ALA on lymphocyte function in SLE. These factors suppress NK function without evidence of lymphocyte cell death or inhibition of NK effector cell binding to tumour targets.     

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways

INTRODUCTION: Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated. METHODS AND RESULTS: All EWS cell lines tested (n=7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis (n=11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro. CONCLUSION: These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.     

Does Immunotherapy for Treatment of Reproductive Failure Enhance Live Births?

Before effective treatment for reproductive failure can be instituted, the cause of the failure must be determined. A search of PubMed was made to identify the published data regarding diagnosis and treatment of reproductive failure. Results were compared with the frequency of antiphospholipid antibodies (APA) in 2995 women with histories of unexplained infertility, recurrent implantation failure, recurrent pregnancy loss, and fertile women. In addition, pregnancy outcomes among 442 women experiencing reproductive failure and elevated NK cell activity after treatment with intravenous immunoglobulin (IVIg) (N = 242) or intralipids (N = 200) were compared. The prevalence of APA was the same among women with the diagnosis of unexplained infertility, recurrent implantation failure, and recurrent miscarriage. Heparin and aspirin are successful in the treatment of elevated APA among women with recurrent miscarriage but not with recurrent implantation failure. IVIg has been successful in the treatment of recurrent miscarriage and recurrent implantation failure among women with elevated APA and/or NK cell activity. When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cell cytotoxicity treated with intralipid were compared with women treated with IVIg, no differences were seen. Immunotherapy for treatment of reproductive failure enhances live birth but only in those women displaying abnormal immunologic risk factors.     

Alterations of rat natural killer (NK) cell cytotoxicity and cytokine production by 3-methylcholanthrene (3-MC)

The carcinogen 3-methylcholanthrene (3-MC) was found to exert immunosuppressive effects both in vitro and in vivo in this study. Spleen cells from 8-week-old male, Sprague-Dawley (S-D) rats exposed to 1, 10 or 100 micrograms/ml 3-MC in vitro for 18 h exhibited a dose-dependent decrease in natural killer (NK) cell cytotoxicity against the YAC-1 tumor target cells in a 4 h 51Cr-release assay. Peritoneal macrophage production of prostaglandin E2 (PGE2) was significantly decreased at all three 3-MC concentrations following a 24 h exposure in vitro. No effect of 3-MC on splenic interleukin-2 (IL-2) production was observed. A separate group of rats was inoculated with a single subcutaneous dose of 5 or 10 mg 3-MC and cytotoxic activity of spleen NK cells was examined at 1, 2, 3, 7, 14, 21, 28, 60, 120 and 180 days after the 3-MC injection. Natural killer cell cytotoxicity was suppressed as early as 24 h after 3-MC injection and persisted up to 21 days. This decrease in NK activity was accompanied by a decreased production of splenic interferon and elevated production of PGE2 by peritoneal macrophages. Natural killer cell cytotoxicity was elevated in the 3-MC-treated rats at 28 and 60 days post-treatment. At 120 and 180 days post-3-MC treatment, when the rats were bearing palpable chemically-induced tumors, NK activity was again significantly depressed. In addition, 3-MC-induced tumors were surgically removed and cultured in vitro. Supernatants from these tumor cell lines were shown to markedly inhibit NK cytotoxicity when tested in vitro. Preliminary results indicate that this inhibition may be mediated by prostaglandins.