Phenotypic and functional studies of leukocytes in human endometrium and endometriosis

The aetiology of endometriosis, a common and disabling disorder, is presently unknown, although immune dysfunction could allow ectopic endometrial fragments to survive outside the uterine cavity. These studies investigate the relationship between leukocyte populations, steroid hormone receptor expression, proliferative activity, bcl-2 expression and apoptosis in eutopic and ectopic endometrium from women with endometriosis or adenomyosis at different phases of the menstrual cycle. Significantly increased oestrogen receptor expression, bcl-2 expression and numbers of CD8+ leukocytes were found in ectopic compared with eutopic endometrium in endometriosis, and CD56+ endometrial granulated lymphocytes (eGLs) were significantly reduced in ectopic endometrium. Apoptotic cells were rarely found in control and subject endometria. In contrast with endometriosis, adenomyotic lesions showed identical steroid hormone receptor expression, proliferative activity, bcl-2 expression and leukocyte subpopulations to eutopic endometrium, indicating different aetiologies for these disorders. The unusual CD56+ CD16- eGLs present in large numbers in late secretory phase eutopic endometrium were highly purified (>98%) by immunomagnetic separation. Except for a negligible cytotoxic activity of eGLs from early proliferative samples, cytotoxic activity of eGLs from non-pregnant endometrium during the menstrual cycle was comparable with those in peripheral blood, predominantly CD56+ CD16+ natural killer cells. eGLs from non-pregnant endometrium and early pregnancy showed a variable proliferative response to 5 and 100 U/ml interleukin-2 over 48-h and 120-h time courses. eGLs are evidently functionally important in the eutopic endometrium. Their absence in endometriotic lesions together with increased CD+8 T-cell numbers and increased oestrogen receptor and bcl-2 expression may have significant effects on the development and progression of endometriosis.     

Intraepithelial leukocytes in endometriosis and adenomyosis: comparison of eutopic and ectopic endometrium with normal endometrium

Intraepithelial leukocytes (IEL) are recognized as an importantcomponent of most mucosal surfaces but have received scant attentionin the human female reproductive tract. The aim of the presentstudy was to characterize, quantify and compare IEL populationsin normal endometrium (n = 30) and in eutopic and ectopic (endometrioticor adenomyotic lesions) endometrium from women with endometriosis(n = 30) or adenomyosis (n = 15) at different menstrual cyclephases in order to assess the role of IEL in these common butpoorly understood disorders. IEL populations were examined informalin-fixed, paraffin-embedded sections using a streptavidin-biotin-peroxidasecomplex technique and quantified in relation to epithelial cellnumbers. IEL in control endometrium and eutopic endometriumin endometriosis and adenomyosis varied during the menstrualcycle, with CD45+, CD43+ and CD56+ cells increasing from theproliferative to the late secretory phase. IEL were elevatedin surface compared with glandular epithelium in the proliferativeand early secretory phases. Throughout the menstrual cycle therewere no significant differences in IEL between eutopic and ectopicendometrium in adenomyosis. Endometriotic foci, however, containedelevated levels of CD45+, CD3+ and CD8+ cells and reduced numbersof CD56+ cells compared with the corresponding eutopic endometriumand these did not vary with menstrual cycle phase. In contrast,ectopic endometrium in adenomyosis showed some cyclical changeswith CD56+ cells increasing significantly in the late secretoryphase. It is possible these differences may play a role in thepathogenesis of endometriosis and the associated complications.     

Immunology: Expression of perforin, granzyme A and TIA-1 by human uterine CD56+ NK cells implies they are activated and capable of effector functions

At the time the human placenta is established, the uterine mucosallining (decidua) is infiltrated by abundant CD3^– CD56^brightnatural killer (NK) cells. NK cells circulating in blood areknown to contain perforin and granzyme A in their cytoplasmicgranules. TIA-1, an RNA-binding protein capable of inducingDNA fragmentation, has also been found in the granules of cytolyticcells. In this paper, we demonstrate the presence of perforin,granzyme A and TIA-1 in the granules of uterine NK cells. Sixteensections of non-pregnant endometrium throughout the menstualcycle and six sections of early decidua, together with cytospinsof four preparations of isolated decidual leukocytes were stainedby both immuno-histology and immuno-electron microscopy to localizeperforin, granzyme A and TIA-1 to the cytoplasmic granules ofCD56⁺ cells. The presence in vivo of these cytolytic moleculesin a normal physiological situation implies that these uterineNK cells may have effector functions in the control of normalplacentation.     

Contraception: Endometrial lymphomyeloid cells in abnormal uterine bleeding due to levonorgestrel (Norplant)

Endometrial lymphomyeloid cell subsets were evaluated in samplesfrom normal women and from women with abnormal uterine bleedingdue to subcutaneous levonorgestrel implants (Norplant) or anintrauterine device (IUD). The frequency of CD3⁺, CD68⁺, CD43⁺and endometrial granulated lymphold cells was evaluated by inimunohistochemicalor phloxine-tartrazine staining of formalinfixed paraffin-embeddedsamples. In normal women, cyclic variation in lymphomyeloidsubsets was seen. In women using Norplant for contraception,the frequency of CD3⁺, CD68⁺ and CD43⁺ cells was dramaticallydecreased, compatible with endometrial atrophy. When Norplantusers with abnormal bleeding were compared to women withoutbleeding, however, the number of CD68⁺ cells was significantlyincreased and the number of CD3⁺ and CD43⁺ cells was preserved,contrary to the hypothesis that this group would show a greaterdegree of atrophy and hence, tissue fragillty. A similar patternwas seen in a preliminary study of women with bleeding associatedwith use of copper-only IUD contraception, and in samples takenfrom late secretory and menstrual phase biopsies from normalcycling women. Whether these changes in endometrial lymphomyeloidcells represent a result of bleeding arising from a common mechanismor rather cause the uterine bleeding is discussed.     

绿色荧光蛋白转基因小鼠CD34+/CD45+细胞在脊髓全横断大鼠模型中的存活及迁移

目的:探讨CD34⁺/CD45⁺细胞移植入脊髓全横断大鼠模型后的存活、迁移及分布情况。方法:体外培养绿色荧光蛋白（GFP）转基因小鼠骨髓细胞，经CD34、CD45单克隆抗体鉴定后移植入脊髓全横断大鼠模型脊髓横断处尾侧，分别在术后24 h、48 h、1周、2周、4周和8周行左心腔内灌注，取出脊髓，连续切片（片厚 10 μm），置于荧光显微镜下观察切片中有无绿色荧光细胞，并观察荧光细胞的分布范围；用免疫组化法检测CD34⁺/CD45⁺细胞的存活。 结果:脊髓横断处头尾两侧均可见绿色荧光细胞，且多分布于灰质中，散在或聚集成片；免疫组化法可见切片中有CD34⁺/CD45⁺细胞散在。 结论: CD34⁺/CD45⁺细胞可在脊髓全横断大鼠模型的脊髓中存活，并可迁移至脊髓横断处头侧，且随时间的延长迁移距离有所增加。     

Isolation of Decidual Lymphocytes From Chorionic Villus Samples: Phenotypic Analysis and Growth in Vitro

PROBLEM : Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. METHOD : Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. RESULTS : A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56^+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4⁺ and CD8⁺ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56^+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56^+bnghtcells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56⁺ cells. CONCLUSION : CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.     

人源化NOD/SCID小鼠免疫细胞的动态变化与鉴定

目的： 比较脐血干细胞与单个核细胞移植NOD/SCID鼠所建立的人源化SCID模型，分析人源化淋巴细胞重建。方法： 磁珠分选法分离脐血中CD34⁺细胞，淋巴细胞分层液分离脐血单个核细胞，分别经尾静脉输入NOD/SCID小鼠。每隔2周采血至10周，流式细胞术动态检测人源淋巴细胞CD45、CD19、CD3抗原。第10周处死小鼠收集外周血、骨髓、胸腺组织，RT-PCR检测模型鼠组织中人β₂M基因及RAG2基因。结果： 两种类型细胞移植均可重建人源免疫细胞，人源淋巴细胞表达水平均在第8周达高峰。骨髓中人源淋巴细胞表达水平明显高于外周血。RT-PCR在外周血与骨髓检测到人β₂M基因及RAG2基因标志。结论： CD34⁺细胞移植重建人源化NOD/SCID免疫系统模型效果要好于脐血单个核细胞。人源T淋巴细胞在模型鼠骨髓中分化成熟。     

Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells

BACKGROUND: Human endometrium is a highly regenerative tissue. We hypothesizedthat the source of endometrial stromal and vascular regenerationis a resident stromal stem/progenitor cell population. Putativehuman endometrial stromal stem/progenitor cells have been identifiedusing clonal assays, a retrospective functional stem cell assay.Therefore, the aim of this study was to screen potential stemcell markers for the prospective isolation of human endometrialstromal stem/progenitor cells and to determine their capacityto identify colony-forming stromal cells. METHODS: Single-cell suspensions of human endometrial stromal cells weresorted using fluorescence-activated cell sorting into positiveand negative populations based on STRO-1, CD133, CD90 or CD146expression for clonal assays. All markers were immunolocalizedin human endometrium. RESULTS: Small populations (2–9%) of human endometrial stromalcells expressed each of the markers. Only CD146⁺ cells wereenriched for colony-forming cells, and CD90^hi cells showed atrend for greater enrichment compared with CD90^lo cells. STRO-1and CD146 were localized to perivascular cells of the endometrium.CD90 was strongly expressed by functionalis stroma and perivascularcells, but only weakly expressed in the basalis stroma. CD133was expressed by epithelial cells of the endometrium, ratherthan by stroma or perivascular cells. CONCLUSIONS: This study identified CD146 as a marker of colony-forming humanendometrial stromal cells supporting the concept that humanendometrium contains a population of candidate stromal stem/progenitorcells.     

Contribution of bone marrow-derived stem cells to endometrium and endometriosis

Bone marrow-derived cells (BMDCs) can differentiate into nonhematopoietic cells, suggesting that BMDCs may contribute to the maintenance of multiple tissues. Donor-derived bone marrow cells have been identified in human uterine endometrium. Here, two murine models were used to investigate the contribution of nonendometrial stem cells to endometrium. We investigate whether BMDCs can localize to uterine endometrium and to endometriosis. After bone marrow transplantation, male donor-derived bone marrow cells were found in the uterine endometrium of female mice. Although uncommon (<0.01%), these cells can differentiate into epithelial cells. After generation of experimental endometriosis by ectopic endometrial implantation in the peritoneal cavity, bone marrow from LacZ transgenic mice was used for transplantation. LacZ expressing cells were found in the wild-type ectopic endometrium implanted in the peritoneal cavity of hysterectomized LacZ transgenic mice. The repopulation of endometrium with bone marrow-derived stem cells may be important to normal endometrial physiology and also may help to explain the cellular basis for the high long-term failure of conservative alternatives to hysterectomy. The examination of a sexually dimorphic organ such as the uterus demonstrates the ability of male bone marrow, which cannot harbor circulating endometrial cells, to generate endometrium de novo and proves their mesenchymal stem cell origin. Finding Y chromosome bearing endometrial cells demonstrates the potential to recapitulate embryonic developmental pathways that were never activated in males; BMDCs may have vast regenerative capacity. Additionally, the ability of stem cells to engraft endometriosis has implications for the origin and progression of this disease. Ectopic differentiation of stem cells may be a novel mechanism of disease. Disclosure of potential conflicts of interest is found at the end of this article.     

Human CD4/CD45RA+ and CD4/CD45RA T cell subsets express CD4-p56lck complexes, CD4-associated lipid kinases, TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates

T cell activation appears to be regulated by an interplay betweenprotein tyrosine kinases (PTKs) and protein tyrosine phosphatases(PTPases). p56^lck and p59^fyn have been found to associate withCD4 and TCR-CD3 respectively. The CD45 family of transmembranePTPases has been shown to be able to regulate the activitiesof these receptor-associated PTKs in vitro. In man, CD45 containsfive different isoforms whose distribution defines subsets ofT cells having distinct activation requirements and in vitrofunctions.Several groups have reported a physical interaction betweendistinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex.Given the potential regulatory interaction between CD45 andPTKs in CD4⁺ subsets expressing different CD45 isoforms, wehave examined CD4 associated and TCR-CD3^– associated PTKactivities, associated phosphatidyl inositoi (PI) kinases andsubstrates of tyrosine phosphoryiation in CD45RA⁺and CD45RA^–CD4⁺ T cell lines derived from peripheral blood. Both subsetsexpress CD4-assoclated p56^lck and TCR-CD3-associated p59^fynkinases which exhibit identical in vitro phosphoryiation atthe Y-394 and Y-420 autophosphorylation sites respectively.Further, both subsets exhibited PI kinases activity associatedwith CD4-p56^lck. Consistent with these observations, anti-CD3crosslinklng induced the phosphoryiation of a similar spectrumof intracellular substrates in these CD45RA⁺and CD45RA^–CD4⁺ T cell lines. These observations indicate that despitethe possible interaction between CD45 isoforms and CD4 or TCR-CD3,the mere expression of the CD45RA isoform does not in and ofitself alter the presence of receptorassociated kinases or theirintracellular targets.     

Activation of CD45-deficient T cell clones by lectin mitogens but not anti-Thy-1

CD45, the leukocyte-common antigen, Is a transmembrane proteintyrosine phosphatase uniquely expressed by cells of hematopoletlcorigin. We have developed CD4⁺ and CD8⁺ T cell clones that aredeficient in the expression of CD45 and have previously shownthat these cells fall to proliferate in response to antigenor cross-linked CD3. These studies have now been extended toshow that stimulation with antl-Thy-1, a mltogenlc signal forthe CD4⁺CD45⁺ and CD8⁺CD45⁺ T cells, falls to induce proliferationin the CD45^– T cells. Examination of the CD8⁺CD45^–T cells correlates antl-Thy-1 unresponslveness with a failureto increase in tyrosine phosphorylatlon. Furthermore, stimulationof CD8⁺CD45⁺ T cells with antl-Thy-1 results in an increasein p56^ick activity but not in CD8⁺CD45^– T cells. In contrastto the results with antl-Thy-1, both the CD4^+– CD45 andCD8⁺CD45^– T cells respond to treatment with lectin mitogens,concanavalln A or phytohemagglutlnln. Lectin-lnduced proliferationwas inhibited by the addition of cyclosporln A. Treatment ofCD45^– T cells with PMA and lonomycln also results in proliferationindicating that activation of protein kinase C in conjunctionwith an increase in intracellular calcium rescues the defectcafsed by CD45 deficiency. The data suggest that CD45 Is requiredfor the activation of tyrosine kinase activity Immediate orprior to transmembrane signaling.     

Characteristic changes of large granular lymphocytes that strongly express CD56 in endometrium during the menstrual cycle and early pregnancy

Large granular lymphocytes that strongly express CD56 (CD56++ LGL) constitute a major population of leukocytes in the secretory endometrium and pregnancy decidua and are considered to be involved in reproductive immunity and in maintaining the pregnancy. The present study aimed to reveal the relationship between the characteristic changes of CD56++ LGL and altered hormonal environment and/or trophoblast invasion in the endometrium. Cell surface markers of CD56++ LGL obtained from the endometrium during the menstrual cycle and early pregnancy were analysed using flow cytometry. The percentages of both CD56++ LGL that express activation antigens (CD69, HLA-DR) and those that express lymphocyte function associated antigen-1 (LFA-1) (CD11a/CD18) were highest in the proliferative phase and decreased gradually throughout the menstrual cycle. Expression of these antigens was further suppressed in the late secretory phase, as well as in the early stage of pregnancy. However, the percentage of CD56++ LGL that express these antigens was significantly higher in spontaneous abortions than in normal pregnancies. On the other hand, the percentage of CD56++ LGL that express CD45RA was lower during normal pregnancy than during the menstrual cycle. The present results suggest that characteristics alterations of CD56++ LGL are regulated by altered hormonal environment and by trophoblast invasion.      

Regulation of chronic colitis in athymic nu/nu (nude) mice

The objective of this study was to assess the roles of NK cells,B cells and/or intraepithelial lymphocytes (IEL) in suppressingthe development of colitis in nude mice reconstituted with CD4⁺CD45RB^highT cells. BALB/c nude mice were lethally irradiated and reconstitutedwith bone marrow from different immunodeficient mice to generateathymic chimeras devoid of one or more lymphocyte populations.Transfer of CD4⁺C45RB^high T cells into chimeric recipients devoidof B cells, T cells and IEL produced severe colitis within 6–8weeks, whereas transfer of these same T cells into B cell- andT cell-deficient or T cell-deficient chimeras produced littleto no gut inflammation. In addition, we found that nude micedepleted of NK cells or RAG-1^–/– mice reconstitutedwith IEL failed to develop colitis following transfer of CD45RB^highT cells. Severe colitis could, however, be induced in nude miceby transfer of activated/T_h1 CD4⁺CD45RB^low T cells. Taken together,our data suggest that IEL, but not B cells or NK cells, playan important role in suppressing the development of chroniccolitis in this model. In addition, our data demonstrate thatsuppression of disease may be due to polarization of naive CD4⁺cells toward a non-pathogenic and/or regulatory phenotype.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V and V_ß regionsat the DNA level in the CD4⁺CD45RO⁺ memory T cell populationof synovial tissue infiltrating T lymphocytes of three rheumatoidarthritis (RA) patients and one patient with chronic arthritis.Cell lines of CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO⁺ andCD8⁺CD45RO^– T lymphocyte populations were generated followingFACS cell sorting of freshly isolated synovial tissue mononuclearcell infiltrates (STMC) and of freshly isolated peripheral bloodmononuclear cells (PBMC) of these patients. The phenotyplc andmolecular analyses have revealed the following. (I) The TCRrepertoires of tissue infiltrating T lymphocytes in the varioussubsets were extensive on the basis of TCR V gene family usage.(II) Furthermore, each patient displayed individual specificTCR V gene expression patterns in the various STMC and PBMCderived T cell subsets. However, the majority of these arthritispatients manifested increased expression of multiple TCR V genefamilies in the synovial tissue derived CD4⁺CD45RO^– Tcell population when compared with the peripheral blood derivedCD4⁺CD45RO⁺ subset. Of these gene families, we found enhancedexpression of the TCR V7 and V_ß11 gene segments insynovial tissue to be shared by all four patients analyzed.OH) Nucleotlde sequence analysis of the CDR3 regions of a numberof TCR V regions in the CD4⁺CD45RO⁺ T cell subsets has revealedthat the CDR3 regions comprised within synovial tissue derivedTCR V regions differed from those found in peripheral bloodderived TCR V regions. These differences in CDR3 diversity mightbe the consequence of a specific interaction with particularMHC-peptlde complexes expressed at the site of inflammation.(Iv) The CDR3 region analysis also showed individual specificamlno acid motifs within the N-D-N regions of all analyzed TCRV_ß genes derived from PBMC as well as STMC.     

Increased Expression of Cell Surface Markers on Endometrial γδ T-Cell Receptor+ Intraepithelial Lymphocytes Induced by the Local Presence of the Sheep Conceptus

PROBLEM: In sheep, γδ T-cell receptor (TCR)⁺ cells are a major lymphocyte subpopulation in the luminal epithelium of the uterine endometrium. During late pregnancy, this population of T cells increases in number and becomes more granulated. This study was performed to determine whether this apparent activation was induced by local effects of the conceptus or systemic effects of pregnancy. METHODS: The unilaterally-pregnant ewe, in which the conceptus is surgically confined to one uterine horn, was used to distinguish between systemic and local effects of pregnancy on function of endometrial γδ TCR⁺ intraepithelial lymphocytes. Lymphocytes collected from peripheral blood, and from the endometrial luminal epithelium of cyclic and unilaterally-pregnant ewes (day 140 of gestation) were analyzed by flow cytometry. RESULTS: As compared to γδ TCR⁺ lymphocytes in peripheral blood, γδ TCR⁺ intraepithelial lymphocytes from non-pregnant ewes had a lower percentage of cells staining positive for CD25, CD44, and L-selectin. There was no effect of pregnancy on the percentage of γδ TCR⁺ peripheral blood lymphocytes staining positive for CD25, CD44, CD29, or L-selectin. Similarly, the percentage of intraepithelial lymphocytes staining positive for these antigens was similar for cells collected from cyclic ewes and cells from the nonpregnant uterine horn of unilaterally-pregnant ewes. In contrast, there was a higher percentage of CD25, CD44, CD29, and L-selectin⁺ cells for γδ TCR⁺ intraepithelial lymphocytes from the conceptus-containing uterine horn of pregnant ewes than for γδ TCR⁺ intraepithelial lymphocytes from the endometrium of cyclic ewes or from the nonpregnant uterine horn of pregnant ewes. CONCLUSION: The local presence of the conceptus causes changes in cell surface marker expression on endometrial γδ TCR⁺ intraepithelial lymphocytes during pregnancy. These changes may reflect conceptus-induced activation of this population of cells.     

Uterine natural killer cells and angiogenesis in recurrent reproductive failure

BACKGROUND: Increased numbers of phenotypically unusual CD56^bright CD16–uterine natural killer (uNK) cells have been associated withrecurrent reproductive failure. uNK cells produce angiogenicgrowth factors and are potential regulators of decidual angiogenesisin early pregnancy. The final common mechanism for early pregnancyloss is thought to be early onset of the maternal circulationand excessive placental oxidative stress. We tested the hypothesisthat increased uNK cells in preimplantation endometrium areassociated with altered angiogenesis. METHODS: Women with recurrent reproductive failure (n = 122) were investigatedwith uterine artery Doppler and endometrial biopsy. Immunohistochemistrywas used to identify uNK, endothelial and vascular smooth musclecells and image analysis was used to assess location, densityand differentiation. RESULTS: uNK cell density was positively correlated with the formationof blood (P = 0.005, r = 0.5) and lymphatic vessels (P = 0.0001,r = 0.6), spiral arteriole smooth muscle differentiation (P= 0.01, r = 0.5) and endometrial oedema (P = 0.004). The functionaleffect of this was a reduced uterine artery resistance to bloodflow. CONCLUSIONS: These data suggest that uNK cells may regulate angiogenesisin non-pregnant endometrium. The mechanisms of reproductivefailure associated with increased uNK cell density appear tobe increased angiogenesis and peri-implantation blood flow,which may lead to early maternal circulation and hence pregnancyfailure due to excessive oxidative stress.     

Immunology: CD56+ lymphoid cells in human first trimester pregnancy decidua as a source of novel transforming growth factor-{beta}2-related immunosuppressive factors

The lymphomyeloid cells isolated from normal first trimesterpregnancy decidua may be separated into a CD56⁺ population ofnatural killer (NK)-lineage cells with the morphology of granulatedlymphocytes, and a CD56^– population which includes othercell types. Unlike CD56+ NK cells in peripheral blood, decidualCD56⁺ cells lack type III Fc receptors (CD16) and did not expresssignificant levels of either type I FcR (CD64) or type II FcR(CDw32). By contrast to the decidual CD56^– cells, CD56⁺cells could release biologically active transforming growthfactor (TGF)-in vitro, detectable using an normal rat kidneyfibroblast colony-forming assay. The CD56⁺ cells could be stainedusing an antibody specific for TGF-2, and similarly stainingcells could be detected in intact biopsies of normal pregnancydecidua. Bioactive TGF- is known to suppress the generationof cytotoxic cells in vitro, and high performance liquid chromatographyfractionation of supernatants conditioned by CD56⁺ but not CD56–cells contained reproducible peaks of immunosuppressive activityat 40–45 and 15–20 kDa, similar to the TGF-2 immunosuppressiveactivity in supernatants conditioned by unfractionated decidua.     

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes

The CAMPATH-1 (CD52) antigen is a 21–28 kDa glycopeptidewhich is highly expressed on lymphocytes and macrophages andis coupled to the membrane by a glycosylphosphatidylinositol(GPI) anchoring structure. The function of this molecule isunknown. However, it is an extremely good target for complement-mediatedattack and antibody-mediated cellular cytotoxicity. The humanizedCAMPATH-1H antibody, which is directed against CD52, is veryefficient at mediating lymphocyte depletion in vivo, and iscurrently being used in clinical trials for lymphoid malignancyand rheumatoid arthritis. It is therefore important to examinethe functional effects of this antibody on different lymphocytesub-populations. Because several other GPI-linked moleculesexpressed on the surface of T lymphocytes are capable of signaltransductlon resulting in cell proliferation, we have investigatedwhether the CAMPATH-1 antigen can also mediate these effects.In the presence of phorbol esters and cross-linking anti-lgantibodies, mAbs specific for CD52 induced proliferation andlymphokine production in highly purified resting CD4⁺ and CD8⁺T lymphocytes. The ret lgG2c YTH 361.10 anti-CD52 antibody,however, was able to activate resting CD4⁺ and CD8⁺ T cellsdirectly without cross-linking or phorbol myristate acetatein the absence of Fc-bearlng cells. Anti-CD52 antibodies alsoaugmented the anti-CD3 mediated proliferatlve response of CD4⁺and CD8⁺ T cells when the two antibodies were co-immobilizedonto the same surface or cross-linked in solution by the samesecond antibody. Both CD4⁺CD45RA and CD4⁺CD45RO T cells werestimulated to proliferate by anti-CD52 antibodies in the presenceof appropriate co-stimulatory factors. Antl-CD52 mAbs did not,however, synerglze with anti-CD2 or CD28 mAb to induce CD4⁺T cell proliferation. The activation of CD4⁺ T cells by antl-CD52antibodies was inhibited by cyclosporin A, suggesting a rolefor the calcineurin-dependent signal transduction pathways.Although CD52 could transduce a signal In T cells, anti-CD52antibodies did not inhibit antigen-specific or polyclonal Tcell responses, suggesting this molecule does not play an essentialco-stimulatory role in normal T cell activation.     

Characterization of Lymphocyte Subpopulations and T Cell Activation In Endometriosis

PROBLEM: Numerous studies have characterized the lymphocyte subpopulations in normal eutopic endometrium and suggested a role for the cytokine secretory products of these lymphocytes in regulating endometrial cell proliferation and differentiation. Recent studies have shown that ectopic endometrium contains a greater concentration of scattered stromal lymphocytes than does eutopic endometrium. However, the lymphocyte subpopulations and their activation status have not been characterized in ectopic endometrium. METHODS: We performed immunohistochemical studies on serial sections of proliferative and secretory phase eutopic endometrium and ectopic endometrium obtained during the proliferative phase using monoclonal antibodies to CD4 (T helper-inducer cells), CD8 (T cytolytic-suppressor cells), CD22 (B-cells), CD56 (natural killer cells), and VLA-1 (T-cell activation marker). RESULTS: Ectopic endometrium contained significantly more scattered stromal CD4, CD8, and activated T cells than did proliferative and secretory eutopic endometrium. There were more activated T-cells in proliferative than in secretory eutopic endometrium. Ectopic endometrium contained significantly fewer NK cells than proliferative and secretory endometrium. CONCLUSIONS: These results demonstrate that (1) the increased lymphocyte population in ectopic endometrium is due to increased numbers of CD4 and CD8 cells, and (2) a greater number of activated T cells are present in ectopic endometrium as compared to eutopic endometrium. Increased concentration of stromal T cells and enhanced VLA-1 expression in ectopic endometrium suggest that cytokine products of the activated T-cells may be involved in regulating cellular processes of endometriosis tissue.