Epithelial cells are the major source of biologically active granulocyte macrophage colony-stimulating factor in human endometrium.

Granulocyte macrophage colony-stimulating factor (GM-CSF) has emerged as an important growth factor for trophoblast and other placental cells, leading to improved placental functioning and fetal survival. Recent observations have indicated that GM-CSF is synthesized by epithelial cells in the murine pregnant and non-pregnant uterus. In this study, the production of GM-CSF by cells derived from human endometrium is assessed using a sensitive bioassay and specific neutralization of the cytokine bioactivity with a monoclonal antibody to GM-CSF. Originally, GM-CSF was assayed in the culture supernatants of explant cultures of human endometria. Concentrations of GM-CSF up to 4440 pg/ml were detected. Subsequently, enriched epithelial cell cultures were prepared from glands isolated from human endometrium. The purity of epithelial cultures was demonstrated by the expression of cytokeratin, a weak immunoreactivity for vimentin and a lack of immunoreactivity for leukocyte common antigen, CD68, a macrophage-specific protein and endothelial marker (factor VIII-related antigens). Detected concentrations of GM-CSF were as high as 18,800 pg/ml. Furthermore, pure epithelial cells of a neoplastic endometrial cell line ECC1 secreted GM-CSF, confirming the ability of endometrial epithelial cells to secrete this cytokine. The immunostaining of dated endometria from proliferative and secretory phases showed primarily that epithelial cells, and to a lesser extent stromal cells, exhibited immunoreactivity for GM-CSF. A Western blot analysis, performed to validate the immunohistochemical data, confirmed the presence of an immunoreactive gene product for GM-CSF in human endometrium throughout the menstrual cycle. These findings indicate that human endometrium synthesizes GM-CSF and that epithelial cells are a major contributor to its production.

Passive acquisition of leukocyte proteins is associated with changes in phosphorylation of cellular proteins and cell-cell adhesion properties.

In this report, we show that interaction of neoplastic epithelial cells with vesicles derived from leukocytes results in passive acquisition by tumor cells of a diverse group of leukocyte proteins. Vesicles shed from leukocytes were heterogeneous and exhibited the specific proteins expressed on leukocyte subsets. Accordingly, epithelial cells differentially acquired leukocyte proteins associated with vesicles. Ultrastructural localization demonstrated that acquired proteins were associated with the plasma membranes of the epithelial cells. The binding of tumor cells that passively acquired leukocyte proteins to immobilized intercellular adhesion molecule-1 and to endothelial cells was significantly increased. Furthermore, passive acquisition of proteins on the plasma membranes of epithelial cells was associated with modulation of overall phosphorylation of proteins in the range of 20-65 kd and consisted of both increased as well as decreased phosphorylation of specific protein species in the cells. These findings demonstrate that leukocyte proteins that are shed in association with vesicles passively coat the plasma membranes of target epithelial cells. Passive acquisition of proteins by cells modulates the constitutive properties endowed upon cells by their native plasma membranes and is associated with changes in phosphorylation of cell proteins.

Extracellular matrix and the patterns of differentiation of human endometrial carcinomas in vitro and in vivo.

Adenocarcinomas differ in their ability to form glandular structures, and the mechanism regulating this architectural differentiation is unknown. In the present study, the patterns of differentiation of two human endometrial carcinomas that differed with respect to their ability to form glands in their original host were studied in monolayer and three-dimensional cultures as well as in xenografts in athymic mice. A moderately differentiated adenocarcinoma of human endometrium, EnCa101, transplanted into nude mice formed tumors indistinguishable from the original neoplasm and secreted mucin. A cell line derived from this tumor, ECC-1, formed monolayers on tissue culture substratum and lost the ability to secrete mucin. However, upon culture within Matrigel, the ECC-1 cells formed glandular structures and secreted mucin. Ultrastructural examination revealed morphological polarity, as evident by intraluminal microvilli and characteristic adhesion structures composed of tight, gap, and desmosomal junctions adjacent to the lumen, and secretory activity. Whereas basal lamina was observed in vivo around glandular cells, epithelial cells were not tethered in vitro with this structure. In contrast, the epithelial cells of a poorly differentiated human endometrial adenocarcinoma, AN3, failed to form glands in nude mice or in Matrigel in vitro. These findings illustrate that gland-forming ability is an intrinsic property of well to moderately differentiated adenocarcinoma cells and that only cells with this inherent potential can be induced to form glands in response to appropriate extracellular signals.

Modulation of HLA-DR expression in epithelial cells by interleukin 1 and estradiol-17 beta.

Both ultrapure human interleukin-1 (IL-1) and Escherichia coli derived recombinant IL-1 alpha and beta consistently induced the expression of major histocompatibility class II (HLA-DR) molecules in a human endometrial and a breast carcinoma cell line. [35S]Methionine incorporation into IL-1 induced, immunoprecipitable HLA-DR molecules demonstrated de novo synthesis of both light and heavy chains of the HLA-DR molecules. Lipopolysaccharide, recombinant interleukin-2 and recombinant interleukin-6 failed to induce HLA-DR expression in these epithelial cells. In contrast to the dramatic effect on HLA-DR expression, IL-1 had no effect on the epithelial cell proliferation. Pretreatment of T47D cells with estradiol-17 beta significantly decreased the IL-1 induced HLA-DR expression, and pretreatment of IL-1 with an IL-1 specific antibody, neutralized IL-1 action. These studies demonstrate that a cytokine (IL-1) and a sex steroid hormone estradiol-17 beta can interact to regulate the expression of HLA-DR molecules in epithelial cells.

Expression of leukocyte adhesion molecules in human endometrium.

In the present investigation the distribution of molecules that are involved in the leukocyte binding was studied in human endometrium. The expression of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA-1), and HLA-DR molecules was studied in 13 proliferative and 10 secretory endometria as well as cultures of endometrial glands and stroma by avidin-biotin complex (ABC) procedure using monoclonal antibodies. The ICAM-1 and HLA-DR molecules were both strongly expressed in the lymphoid and endothelial cells. ICAM-1 expression was uniform in the epithelium, whereas the HLA-DR molecules were preferentially expressed in the epithelial cells in the basalis. Expression of both epithelial HLA-DR and ICAM-1 molecules was enhanced adjacent to lymphoid aggregates. ICAM-1 molecule was uniformly expressed in the stromal cells in the basalis and the functionalis, whereas the HLA-DR molecules were expressed exclusively in the stromal cells surrounding the lymphoid cells. ICAM-1 expression in the epithelial and stromal cells was confirmed in the isolated intact stromal cells and glands by immunohistochemistry. Although stromal and epithelial cells propagated in vitro expressed ICAM-1, they were rarely HLA-DR positive. The expression of LFA-1 was confined to the lymphoid cells. The high level of expression of ICAM-1, LFA-1, and HLA-DR molecules in human endometrial constituents may contribute to the presence, aggregation, and preferential distribution of lymphoid cells in human endometrium.

Interleukin-6 immunoreactivity in human tumors.

The cytokine, interleukin-6 (IL-6), has emerged as a likely mediator of many of the systemic alterations observed in patients with cancer (fever, increased erythrocyte sedimentation rate, and alterations in plasma protein composition) and may also mediate local effects such as alteration in proliferation of tumor cells, increased tumor cell motility, and decreased intercellular adhesions between tumor cells. The distribution of IL-6 immunoreactivity in different human tumors was studied. IL-6 immunoreactivity was detected by the avidin-biotin-complex (ABC) procedure using a polyclonal rabbit antiserum raised against an E coli-derived human IL-6 (rIL-6). Preimmune rabbit serum used as a control did not yield specific staining and preadsorption of the IL-6 antiserum with rIL-6 abolished specific staining. Strong-to-moderate IL-6 immunoreactivity was observed in the neoplastic elements present in primary squamous cell carcinomas, in adenocarcinomas of mammary, colonic, ovarian, and endometrial origin, in various adenocarcinomas metastatic to lymph nodes, and in soft tissue tumors including leiomyosarcoma and neurofibrosarcoma. Weak-to-moderate IL-6 immunostaining was observed in Hodgkin's and non-Hodgkin's lymphomas. This study demonstrates that most human tumors stain positively for IL-6, adding weight to the hypothesis that IL-6 is a key cytokine that participates in the host-tumor interaction.

Sex steroid receptors in lymphoid cells of human endometrium.

Sex steroid hormone action on target tissues is mediated through binding of estrogen and progesterone to specific intranuclear proteins, the estrogen and progesterone receptors (ER and PR). Therefore, in the present report the authors investigated for the presence of ER and PR in lymphoid cells of endometrial stroma that may serve as potential targets for estrogen- and progesterone-mediated effects in endometrium. The presence of ER was shown in nine proliferative and ten secretory endometria and the presence of PR in three secretory and one proliferative endometria. ER and PR were localized by monoclonal antibodies, to the nuclei of cells, with the use of, respectively, peroxidase-antiperoxidase (PAP) and avidin-biotin-peroxidase complex (ABC) methods. Lymphoid cells were then delineated by decoration of their plasma membranes with the use of monoclonal antibodies to HLA-DR, leukocyte common antigen (LCA), Leu-4 (CD3), and IL-2 receptor molecules with the use of an ABC staining procedure. A group of cells in the lymphoid aggregates in endometrial stroma showing membranous staining for HLA-DR, LCA, and Leu-4 molecules had nuclear ER. IL-2 receptor-positive cells were rare in endometrium, and no PR-positive cells were found in lymphoid aggregates. Furthermore, HLA-DR and ER were expressed in the glandular and surface epithelium in the proliferative phase and in occasional glands in the basalis in the late secretory phase. The presence of an ER-positive lymphoid cell population in endometrial lymphoid aggregates suggests that these cells may serve as target cells for estrogen.

Cytokine-induced production of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17 beta.

The cytokine IFN-beta 2/IL-6 has emerged as an important means of communication between cells--both within the immune system as well as outside it. In exploring the link between the endocrine and the immune systems, we have studied the secretion of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells and its modulation by estrogens. Endometrial stromal cells produced IFN-beta 2/IL-6 in response to other inflammation-associated cytokines such as IL-1 alpha or beta, TNF, and IFN-gamma. This secretion was strongly inhibited by estradiol-17 beta at concentrations as low as 10(-9) M. Multiple species of stromal cell IFN-beta 2/IL-6 in the size range 23 to 30 kDa were detected using immunoprecipitation or immunoblotting procedures. The endometrial stromal cell IFN-beta 2/IL-6 species were phosphorylated and differentially glycosylated in a manner comparable to IFN-beta 2/IL-6 secreted by induced human peripheral blood monocytes or foreskin fibroblasts. However, in contrast to peripheral blood monocytes and fibroblasts, bacterial LPS did not induce IFN-beta 2/IL-6 production in endometrial stromal cells. Additionally, the IFN-beta 2/IL-6 identified in medium from IL-1 alpha-induced stromal cells is biologically active on hepatocytes. These observations, taken together with the observation that IFN-beta 2/IL-6 strongly inhibits the proliferation of human epithelial cells, suggest the possibility that stromal cell secreted IFN-beta 2/IL-6 may affect the physiology of the overlying epithelium in an hormonally modulated manner. Estrogen-regulated production of endometrial IFN-beta 2/IL-6 may participate in gender-specific systemic immunomodulation.

Eosinophilic granuloma of the cavernous sinus and orbital apex in an HIV-positive patient.

A 35-year-old HIV-positive woman with painful ophthalmoplegia, sensory loss extending to all branches of the trigeminal nerve, and progressive optic neuropathy was found to have eosinophilic granuloma of the cavernous sinus, superior orbital fissure, and orbital apex. There was no radiologic evidence of a lytic bone lesion within the skull or orbit and clinical evidence suggested a primary intracranial origin for this lesion. This is the first case of a cavernous sinus syndrome caused by eosinophilic granuloma and the first time HIV infection is reported in association with histiocytosis-X.

Application of a quick immunoenzymatic labeling as an adjunct to frozen-section diagnosis.

Immunoenzymatic labeling is not currently used in the frozen-section diagnosis of tumors, in view of the length of the procedure. The authors tested whether, without loss of specificity and sensitivity, the labeled avidin-biotin (LAB) method may be adequately shortened for use in frozen-section diagnosis. Cryostat sections of tumors of various types and different degrees of differentiation and a panel of monoclonal antibodies to various molecular weight cytokeratins, vimentin, and leukocyte common antigen was used. The entire LAB procedure was completed in less than 7 minutes. For comparison, all tumors were also stained by the conventional avidin-biotin complex (ABC) method, which required more than one and a half hours for completion. The pattern and intensity of specific staining for the two procedures was identical, and the background staining was minimal with the LAB method. In view of the short time required to obtain specific staining, the quick LAB method may be of great value for characterization of primary or metastatic tumors in frozen-section diagnosis.

Differential expression of HLA-DR, HLA-DP, and HLA-DQ antigenic determinants of the major histocompatibility complex in human endometrium.

We reported on the expression of HLA-DR molecules of the major histocompatibility complex in human endometrium. We now report on the expression of two other class II molecules, the HLA-DP and HLA-DQ determinants. These molecules were localized by monoclonal antibodies in 11 proliferative and 12 secretory endometria by avidin-biotin-complex (ABC) procedure. The expression of all three molecules was invariable and consistent throughout the entire menstrual cycle in endothelial and lymphoid cells. HLA-DR molecules were expressed in endometrial epithelium, particularly in the basalis in the mid to late proliferative phases of the cycle. In contrast, through the entire menstrual cycle, the expression of HLA-DP and HLA-DQ antigenic determinants was absent, and only occasionally a focal expression of these molecules was seen in endometrial epithelium. The in vitro induction of expression of the class II molecules in human endometrial epithelial cell cultures (HEE) by IFN-gamma was dose-dependent. After treatment with low doses of IFN-gamma, these cells primarily expressed HLA-DR molecules in vitro. The differential expression of the molecules of the major histocompatibility complex in human endometrial epithelium in vivo may be due to the differential sensitivity of th epithelium in response to cytokine(s).

Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro.

Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles. To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating. HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells. The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days. HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating. Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface. Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells. The HBV producing cells did not show any evidence of a cytopathic effect. These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo. Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.

Histologic, immunofluorescence, and electron microscopic study of infectious process in mouse lung after intranasal challenge with Coxiella burnetii.

A histologic, immunofluorescence, and electron microscopic study of the intracellular parasitism of Coxiella burnetii (the Q fever agent) in mouse lungs after intranasal challenge was undertaken. It was shown that this microorganism invades type I and, rarely, type II pneumocytes as well as pulmonary fibroblasts and histiocytes. The infectious process can be described as a focal intra-alveolar inflammation with the macrophages prevailing in the exudate. It is self-limited, with a complete resolution. The inflammation is associated with atelectases and with increased secretory activity by type II pneumocytes. Alveolar macrophages and granulocytes degrade C. burnetii. This degradation is followed by damage to and eventual disintegration of some macrophages and by damage to some bacterium-free pneumocytes and vascular endothelial cells in the vicinity of macrophages degrading organisms. The cell damage might be caused by lipopolysaccharide released from degraded organisms. The infectious process is also associated with the influx of T cells in the pneumonic foci, T-cell attachment to the macrophages degrading organisms, and fusion of some macrophages. These are considered a morphologic expression of cell-mediated immunity involved in the infectious process.

Antiproliferative effect of interferon-gamma in human endometrial epithelial cells in vitro: potential local growth modulatory role in endometrium.

We previously reported that recombinant interferon-gamma (IFN-gamma) induces HLA-DR (human lymphocyte antigen) molecules of the major histocompatibility complex in human endometrial epithelial cells in vitro. We now report that IFN-gamma inhibits the proliferation of human endometrial epithelial cells and a human endometrial carcinoma cell line (EnCa101AE). Human endometrial epithelial cells expressed HLA-DR molecules and underwent morphological changes when exposed to IFN. Furthermore, the proliferation of these cells, as evidenced by nuclear labeling of bromodeoxyuridine (an analog of thymidine that is incorporated into cells in S phase), was markedly reduced, in a dose-dependent manner, by IFN-gamma. IFN-gamma induced HLA-DR expression, morphological changes, shedding from the substratum, and cell death in EnCa101AE cells. In addition, cell number and the numbers of bromodeoxyuridine-, Ki-67 (a nuclear marker of proliferation)-, and MPM-2 (a marker of mitotic cells)-positive cells were markedly lower in the EnCa101AE cultures treated with IFN-gamma than those in control cultures. The cytostatic and HLA-DR-inducing effects of IFN-gamma could be abrogated by neutralization with a polyclonal antibody, and IFN-gamma effects were reversible within days after its withdrawal. These findings indicate that IFN-gamma inhibits proliferation of human endometrial epithelial cells and suggest that this factor may locally regulate the proliferation of these epithelial cells in vivo.

Immunohistochemical distinction of Paget's disease from Bowen's disease and superficial spreading melanoma with the use of monoclonal cytokeratin antibodies.

The differentiation of Paget's disease from Bowen's disease and Pagetoid superficial spreading melanoma may represent diagnostic difficulties. The special stains used in their differential diagnosis are nonspecific and not always sensitive. Therefore, the expression of cytokeratins of different molecular weights (54, 57, and 66 kilodaltons [kD]) was studied in 26 intraepithelial neoplasms in formalin-fixed paraffin-embedded tissues with the use of an avidin-biotin complex (ABC) method with monoclonal cytokeratin antibodies. These included 9 cases of Paget's disease, 11 cases of Bowen's disease, and 6 cases of Pagetoid superficial spreading melanoma. Paget cells from vulva and breast were always positive for 54-kD cytokeratin, variable for 57-kD cytokeratin, and negative for 66-kD cytokeratin. The neoplastic cells in all 11 cases of Bowen's disease were stained for 57-kD and 66-kD cytokeratins but not for 54-kD cytokeratin. The neoplastic cells in all cases of melanoma did not express any of the cytokeratins studied. The results indicate that antibodies to cytokeratins of different molecular weights may be used as a diagnostic tool in the distinction of Paget's disease from Bowen's disease and melanoma.

Comparison of cytokeratin expression in primary and metastatic carcinomas. Diagnostic application in surgical pathology.

Metastatic poorly differentiated carcinomas often represent diagnostic difficulties in surgical pathology. Therefore, the expression of cytokeratins of different molecular weights (54, 57, and 66 kd) were compared in paraffin sections of 37 primary carcinomas with their lymph node metastases by an avidin-biotin complex (ABC) method, using monoclonal antibodies. The epithelial tumors consisted of 16 squamous cell carcinomas (SCCs) and 17 adenocarcinomas with different degrees of differentiation (well, moderately, or poorly differentiated), a renal cell carcinoma, a hepatocellular carcinoma, a transitional cell carcinoma of the bladder, and a carcinoid tumor of the stomach. The primary and metastatic tumors showed the same cytokeratin profiles. All SCCs and their metastases were positive for 57-kd cytokeratin and negative for 54-kd cytokeratin. All adenocarcinomas and their metastases were positive for 54-kd cytokeratin and negative for 66-kd cytokeratin. The extent of reactions varied with the differentiation of the carcinomas, with well-differentiated tumors showing more diffuse staining. Cases of lymphoma, sarcoma, and melanoma were negative for the three types of cytokeratins. The results indicate that identification of different molecular weight cytokeratins may be used to distinguish poorly differentiated SCCs from poorly differentiated adenocarcinomas, even in metastatic tumors. In addition, demonstration of these cytokeratins is useful in substantiating presence and identity of small foci of metastases in lymph nodes.

Immunoultrastructural localization of Ia antigens in human endometrium.

The distribution of Ia antigens was studied at the light and ultrastructural levels in 34 proliferative and 16 secretory endometria with two monoclonal antibodies using an avidin-biotin-peroxidase complex method. The endothelial cells, many lymphocytes, and various monocytic-macrophagic cells in the endometrial stroma were Ia positive. Furthermore, Ia antigens were localized to normal endometrial epithelium in the proliferative phase, and focally in epithelium adjacent to stromal lymphoid aggregates throughout the cycle. Expression of Ia antigens in the secretory epithelium was focal or absent. At the ultrastructural level, Ia-positive epithelial cells exhibited staining on the plasma membrane, in free and membrane-bound ribosomes, rough endoplasmic reticulum, and occasional perinuclear cisternae. In the endothelial cells and lymphocytes, Ia antigens were also localized to the plasma membrane and rough endoplasmic reticulum. These findings indicate that endometrial epithelium expresses Ia antigens which may be regulated by endometrial lymphoid cells and/or hormones. The plasma membrane expression of Ia antigens by the epithelial cells of endometrium appears to result from active synthesis, and not merely from passive absorption of Ia antigens.

Induction of HLA-DR antigen expression in human endometrial epithelial cells in vitro by recombinant gamma-interferon.

Recent studies suggest that HLA-DR antigens may be inducible in some normal epithelial cells. Therefore, we studied the expression of these molecules in epithelial cell cultures derived from 9 proliferative and 7 secretory endometria with and without treatment by recombinant gamma-interferon (gamma-IFN). A panel of monoclonal antibodies to HLA-DR antigens, T cells, B cells, macrophages, endothelial cells, and cytokeratin was used to stain the cultured cells by the avidin-biotin complex method. The cultures consisted only of epithelial cells as demonstrated by expression of cytokeratin and by electron microscopy and did not contain any T or B cells, macrophages, or endothelial cells. The endometrial epithelial cells were HLA-DR-negative under routine culture conditions but expressed HLA-DR antigens after gamma-IFN treatment in a dose- and time-dependent fashion. The findings indicate that endometrial epithelium can actively synthesize HLA-DR antigens in vitro after induction by gamma-IFN.

Variable expression of Ia antigens in human endometrium and in chronic endometritis.

Recent studies suggest that Ia antigens may be expressed in epithelial cells and that their expression may be under hormonal control. Therefore, the distribution of these antigens was studied in frozen sections of 37 human endometria with two monoclonal antibodies (Mab) to monomorphic determinants of Ia antigens using an avidin-biotin-complex (ABC) method. Five early proliferative, 9 midproliferative, 3 late proliferative, and 12 secretory endometria were examined. Two gestational endometria and six endometria with chronic endometritis were also used. Four consecutive sections from each case were stained for Ia, OKT8, Leu-3a, and B1 antigens. Throughout the cycle, the endothelial cells, many lymphocytes, and various monocytic-macrophagic cells in endometrial stroma were Ia positive. Furthermore, Ia antigens were localized to the normal endometrial epithelium. The intensity and the pattern of Ia expression, however, varied in different phases of the cycle. Ia antigens were stained weakly in endometrial glands and surface epithelium in early proliferative phase, and strongly in surface epithelium and glandular cells of the basalis and to a lesser extent of the functionalis in midproliferative and late proliferative phases. The expression of Ia antigens in epithelium was absent or focal during the secretory phase and in gestational endometria. Throughout the cycle and in gestational endometria, glandular cells in intimate association with lymphocytic aggregates were Ia positive. In chronic endometritis, the increased number of Ia positive stromal lymphoid cells was associated with a strong display of Ia antigens in epithelium. The findings indicate that, in addition to endothelial and lymphoid cells, Ia antigens are expressed in endometrial glandular and surface epithelial cells. This expression may be influenced by lymphoid cells in endometrium and by hormones.

Immunohistologic analysis of lymphoid cells by a rapid double staining method.

The development of monoclonal antibodies has allowed characterization of subpopulations of lymphoid cells and of their in situ distribution in tissues. The feasibility of simultaneous localization of two surface antigens was studied by double staining with monoclonal antibodies to B cells, T cell subsets and follicular dendritic reticulum cells (DRC) using the avidin-biotin-complex (ABC) method in sections of human lymphoid tissues (tonsil, lymph node, spleen) and a non-lymphoid tissue, endometrium. Color mixture was avoided when an additional incubation with avidin-biotin-labeled peroxidase and subsequent development in the respective substrate of the first sequence preceded the second staining sequence using the primary antibodies at optimal concentrations. The antigenic profiles were portrayed by contrasting and distinct colors of the reaction products. It was observed that T lymphocytes of the cytotoxic/suppressor and helper/inducer phenotypes were topographically associated with each other and with B cells in B and T cell areas of lymphoid tissues as well as in lymphocytic aggregates in endometria. Subpopulations of these cells were mantled by processes of DRCs in lymphoid follicles. The findings indicate that the double ABC staining method can be used for simultaneous demonstration of two surface antigens in tissue sections.