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.     

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.     

HLA-Cw7 Zygosity Affects the Size of a Subset of CD158b+ Natural Killer Cells

Individuals with certain HLA class I genotypes are highly susceptible to disease after viral infection. Natural killer (NK) cells kill virus-infected cells through a mechanism involving HLA class I receptors. These facts may be connected if an individual's HLA genotype regulates the number and function of NK cells. We have observed that subjects homozygous for the HLA-B/C region of conserved major histocompatibility complex (MHC) extended haplotypes have lower NK cell activity and a significantly lower frequency of CD16⁺CD56⁺ NK cells than heterozygotes. The proportion of CD16^–CD56⁺ NK cells was unaffected by zygosity for the HLA-B/C region. We show here that the frequency of CD16⁺CD158b⁺, but not CD16^–CD158b⁺ NK cells, was significantly lower (p <0.026) in homozygotes for HLA-Cw7 (NK1 ligand) haplotypes than in heterozygotes. The frequencies of CD16⁺CD158a⁺ and CD16^–CD158a⁺ and CD16^–CD158a⁺ or CD16⁺NKB1⁺ and CD16^–NKB1⁺ NK cells were not different in these donor groups. These findings suggest that the proportion of NK cells coexpressing CD16 and CD158b, but not CD158a nor NKB1, is influenced by zygosity for the HLA-Cw7 (NK1 ligand) haplotype. Since NK cells are involved in protection from virus infection, a reduced size of a ligand-specific NK subset in individuals homozygous for some HLA-B/C haplotypes may help explain their increased susceptibility to virus-induced diseases.     

Suppressed Cell-Mediated Immunity and Monocyte and Natural Killer Cell Activity Following Allogeneic Immunization of Women with Spontaneous Recurrent Abortion

Spontaneous recurrent abortion (SRA) has been treated by means of immunization with paternal or third-party white blood cells, yet the immunological basis for SRA and for the role of immunization protocols in pregnancy outcome remains controversial. To elucidate this question, nine women with SRA were immunized with paternal mononuclear cells and studied before and 2 weeks after immunization. Seven women who became pregnant gave birth to live newborns. Secretion of the T helper 1 cytokines IL-2 and interferon- by patients' mononuclear cells decreased, while production of IL-10 increased. The levels of natural killer and lymphokine-activated killer cell-mediated cytotoxicity were markedly decreased. Monocyte functions such as secretion of IL-l, tumor necrosis factor a, IL-6, and cytotoxic activity decreased concurrently with elevations in IL-10 and transforming growth factor secretion. Production of IL-12, a pivotal regulatory cytokine, decreased. Furthermore, B7/1 expression on patients' mononuclear cells was downregulated. This resulted in a decrease in monocyte costimulatory activity of purified T cells with soluble anti-CD3, paralleled by a decline in allogeneic proliferative responses. These results suggest that the improved pregnancy success rate in women with SRA following immunization may be partly related to suppression of cell-mediated immunity and monocyte and natural killer cell activity.     

Intravenous Immunoglobulin Therapy for Recurrent Spontaneous Abortion: A Meta-Analysis

PROBLEM: Published randomized trials of the use of intravenous immunoglobulins (IVIG) as a treatment for recurrent spontaneous abortion (RSA) have produced conflicting results. The purpose of this study was to conduct a systematic review of the current evidence to evaluate the effectiveness of IVIG for RSA. METHOD OF STUDY: After a thorough search of the literature, four randomized, doubleblind trials comparing IVIG with placebo for treatment of RSA were included in the metaanalysis. Live birth rates for each treatment group were extracted, and the overall odds ratio (OR) and absolute treatment effect for IVIG were calculated. RESULTS: Two of the trials showed an increase in successful pregnancy outcome with IVIG treatment and two did not. The overall OR was 1.48 (95% CI, 0.84–2.60) in favor of IVIG, with an absolute treatment effect of 10.1% (95% CI, ?4.8–24.6). Excluding pregnancy failures with an obvious cause produced statistically significant results, but this approach may be subject to bias. CONCLUSION: This meta-analysis suggests that IVIG may have a role in the treatment of recurrent abortion, but as yet no conclusive evidence is available.     

Changes in Endometrial Natural Killer Cell Expression of CD94, CD158a and CD158b are Associated with Infertility

Problem  Cycle-dependent fluctuations in natural killer (NK) cell populations in endometrium and circulation may differ, contributing to unexplained infertility.
Method of study  NK cell phenotypes were determined by flow cytometry in endometrial biopsies and matched blood samples.
Results  While circulating and endometrial T cell populations remained constant throughout the menstrual cycle in fertile and infertile women, circulating NK cells in infertile women increased during the secretory phase. However, increased expression of CD94, CD158b (secretory phase), and CD158a (proliferative phase) by endometrial NK cells from infertile women was observed. These changes were not reflected in the circulation.
Conclusion  In infertile women, changes in circulating NK cell percentages are found exclusively during the secretory phase and not in endometrium; cycle-related changes in NK receptor expression are observed only in infertile endometrium. While having exciting implications for understanding NK cell function in fertility, our data emphasize the difficulty in attaching diagnostic or prognostic significance to NK cell analyses in individual patients.     

Decidual Natural Killer Cytotoxicity Decreased in Normal Pregnancy but Not in Anembryonic Pregnancy and Recurrent Spontaneous Abortion

PROBLEM : The natural killer (NK) cell activity is depressed in the decidua of early normal pregnancy. Recently Morii et al. (Am J Reprod Immunol 1993;29:1–4) found that all early intradecidual CD3⁺ T cells expressed either T cell receptor (TCR) α/β or γ/δ but that the expression of the CD3⁺/TCR complex was down-regulated. METHOD : To test whether these changes in decidual cellular immunity are different among normal pregnancy, anembryonic pregnancy and recurrent spontaneous abortion, we examined the immune cell subpopulations in the decidua from these three types of pregnancy using flow cytometry and an NK cytotoxicity assay. RESULTS : Intradecidual CD3⁺ T cells expressed either TCR α/β or γ/δ, and the level of expression of the CD3/TCR complex was down-regulated in normal pregnancy, anembryonic pregnancy, and recurrent spontaneous abortion. Although the relative proportion of decidual NK cells was increased to approximately the same extent in all three types of pregnancy, decidual NK activity was higher in anembryonic pregnancies and in recurrent spontaneous abortions than it was in normal pregnancies. CONCLUSION : Decidual NK cell responses are different in anembryonic pregnancies and in recurrent spontaneous abortions than in normal pregnancies. Whether this difference is pathogenic or is the response to a dead embryo remains to be elucidated.     

Intravenous Immunoglobulin in the Prevention of Recurrent Spontaneous Abortion: The European Experience

PROBLEM: Clinical trials of the use of intravenous immunoglobulin (IvIg) in the treatment of recurrent spontaneous abortion (RSA) in Europe were reported and discussed. METHOD OF STUDY: A search of the published literature, combined with our own published and unpublished results, was performed. RESULTS: Two placebo-controlled trials have been concluded in Europe. One trial found high but equal success rates in both the IvIg and the placebo group. Another trial found that IvIg treatment may increase the success rate by 24% compared with placebo but that the result was not statistically significant, which might be due to the small number of patients. Reasons for the diverse results of the two trials were discussed. CONCLUSION: More and larger placebo-controlled trials of IvIg treatment in RSA are needed before drawing definitive conclusions.     

补肾中药对溴隐亭致流产鼠模型中蜕膜CD56~+NK细胞表面CD69和CD94表达的影响

为了观察补肾中药是否能影响蜕膜CD5 6 + NK细胞比例及其表面CD6 9、CD94的表达 ,从而进一步阐明补肾中药对母胎界面免疫的影响机制。SD大鼠于孕 6～ 8d皮下注射溴隐亭 0 3mg/kg·d ,建立改良的溴隐亭致SD大鼠流产模型 ,随机分为三组。A组为模型组 ;B、C组在孕 1～ 1 1d分别给予大剂量中药 (4 5g/kg·d)、P(8mg/kg·d) ,另设正常孕鼠对照 (D组 )。孕 1 2d处死 ,取蜕膜组织 ,分离成单个细胞悬液 ,用流式细胞分析技术观察 4组蜕膜CD5 6 + NK细胞的量及蜕膜NK细胞表面CD6 9、CD94的表达差异。结果发现 :B组 (P <0 0 5 )、C、D组 (P <0 0 1 )妊娠率与A组相比增加 ,并且有差异 ;B、C组蜕膜CD5 6 + NK细胞比例与A组相比明显增加 ,与D组无差异 ;各组蜕膜CD5 6 + NK细胞表面CD6 9表达无差异 ;B、C、D组CD94的表达与A组相比显著增加 ;A组蜕膜CD5 6 + NK细胞表达CD6 9与CD94相比明显增加 ,B、C组相反。因此 ,补肾中药能影响蜕膜CD5 6 + NK细胞表面CD6 9、CD94的表达 ,可能通过增加CD94的表达 ,抑制CD6 9从而进一步抑制NK细胞的活性 ,起到保胎作用     

Suppression of Natural Killer Cell Activity by Monocytes Following Immunotherapy for Recurrent Spontaneous Aborters

PROBLEM : Natural killer (NK) cell activity has previously been shown to decrease in normal pregnancy as compared with the nonpregnancy state. The purpose of this study was to determine NK cell activity in recurrent aborters and to investigate the kinetics of NK cell activity following immunotherapy. METHODS : Recurrent aborters (N = 17) were immunized with husbands' mononuclear cells (1 × 10⁸) twice during the early stage of current pregnancy. NK cell activity of recurrent aborters as well as that of normal pregnant (N = 12) and nonpregnant (N = 6) women (controls) was determined by ⁵¹Cr release assay. Monocytes were depleted from the mononuclear cell fraction and its effect on the NK cell activity was determined as well. RESULTS : At around 5 wk of gestation, NK cell activity in recurrent aborters before treatment was significantly higher (28.0 ± 5.1%) than that in normal pregnancy (18.9 ± 4.3%) (P < 0.01). Following immunotherapy, NK cell activity of recurrent aborters (N = 13) who maintained their pregnancy decreased significantly (21.7 ± 8.9%) (P < 0.05). In contrast, NK cell activity of recurrent aborters (N = 4) who aborted their current pregnancy did not decrease. Depletion of monocytes resulted in a significant increase in NK cell activity (P < 0.05). CONCLUSIONS : This study suggests that the immunotherapy induces suppression of NK cell activity which may contribute for the maintenance of pregnancy. Moreover, monocytes may be involved in this suppression.     

Molecular characterization of human CD94: A type II membrane glycoprotein related to the C-type lectin superfamily

Natural killer (NK) cells preferentially express several genes of the C-type lectin superfamily which have been implicated in the regulation of NK cell function. We demonstrate that CD94 is a type II membrane protein encoded by a unique gene of the C-type lectin superfamily. While homology of CD94 with the NK cell-associated NKR-P1 and NKG2 C-type lectin genes is limited to the structural motifs conserved in the carbohydrate recognition domain, all of these genes are on human chromosome 12, the syntenic of mouse chromosome 6, where genes of the NK complex (NKR-P1 and Ly-49) are located. An unexpected feature of CD94 is the essential absence of a cytoplasmic domain, implying that association with other receptors may be necessary for the function of this molecule.     

CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

Up-Regulated Expression of CD56+, CD56+/ CD16+, and CD19+ Cells in Peripheral Blood Lymphocytes in Pregnant Women With Recurrent Pregnancy Losses

PROBLEM : To analyze immunophenotypic profiles of peripheral blood and humoral autoimmune responses in women with a history of recurrent spontaneous abortions (RSA). METHOD : Peripheral blood lymphocyte subsets by flow cytometry and autoantibodies to phospholipids and nuclear components by ELISA were measured in non pregnant and pregnant women with RSA of unknown etiology. Thirty-five pregnant and eighty-one nonpregnant women with RSA were studied. Seventeen nonpregnant and twenty-two pregnant normal controls were included. RESULTS : Natural killer (NK) cells (CD56+) were significantly elevated in nonpregnant women with RSA as compared with nonpregnant controls. Pregnant women with RSA demonstrated significantly increased NK (CD56+, CD56+/CD16+) and B cells (CD19+) as compared with pregnant controls. Women who miscarried the index pregnancy demonstrated significantly lower CD3+ cells in comparison with normal controls. Women with RSA and antiphospholipid antibodies showed significantly elevated NK cells when compared with women without antiphospholipid antibodies. Women with autoantibodies to nuclear components demonstrated significantly elevated CD19+/CD5+ cells when compared to women without autoantibodies to nuclear components. CONCLUSIONS : Women with RSA demonstrate an abnormal cellular immune response by increasing peripheral natural killer cells and B cells as compared with normal controls.     

Molecular characterization of a gene in the rat homologous to human CD94

Three classes of major histocompatibility (MHC) class I binding receptors on natural killer (NK) cells have so far been described: CD94/NKG2 heterodimeric receptors and killer cell inhibitory receptors in the human, and Ly-49 homodimers in rodents. CD94, NKG2 and Ly-49 belong to the C-type lectin super-family. As yet, CD94 and NKG2 molecules have not been detected in rodents or Ly-49 in humans. It has therefore been proposed that the two receptors represent functional equivalents in these species. The present study describes the cDNA cloning of a novel rat gene encoding a protein of 179 amino acids, 54.2 % identical to human CD94. The single-copy Cd94 gene is localized to the rat NK gene complex (NKC), within 50 kb from Nkrp2, between the Nkrp1 and Ly49 gene clusters. By Northern blot analysis, we showed that rat CD94 is selectively expressed by NK cells and a small subset of T cells, similar to the human ortho-logue. This expression is strain dependent, with high expression in DA NK cells and low in PVG NK cells. Evidence is presented that this difference is not due to receptor repertoire shaping by MHC-encoded ligands, but is controlled by genetic elements residing within the NKC. The identification of a rat CD94 orthologue suggests that NK cell populations utilize two different C-type lectin receptors for MHC class I molecules in parallel.     

Women with Pre-Eclampsia Have an Altered NKG2A and NKG2C Receptor Expression on Peripheral Blood Natural Killer Cells

Problem  Preeclampsia, a pregnancy disorder, is associated with exaggerated inflammation and increased serum monokines. Uterine natural killer (NK) cells are implicated in preeclampsia pathology, but little is known regarding peripheral NK cells in the disease.
Method of Study  We examined blood NK cells at delivery in women with preeclampsia, in healthy pregnant women and in healthy non-pregnant blood donors as a reference.
Results  Although the percentages of both NKG2A- and NKG2C-positive NK cells were normal in preeclamptic women, the levels of NKG2A and NKG2C on NK cells were significantly up-regulated in these women. In vitro stimulation of PBMCs from healthy pregnant women and blood donors with monokines resulted in increased percentage of NKG2A⁺ NK cells and increased NKG2A levels, while levels of NKG2C were decreased.
Conclusions  Our results suggest that the peripheral NK-cell pool is skewed in preeclampsia and possibly under the influence of monokines like interleukin (IL)-15 and IL-12.