The optimal application of forward and ninety-degree light scatter in flow cytometry for the gating of mononuclear cells

Peripheral blood mononuclear cells from ten normal donors were labeled with a monoclonal antibody specific for monocytes and analyzed using a fluorescence activated cell sorter (FACS). Forward and 90 degrees light scatter parameters were studied in order to apply optimal computerized gating to identify and exclude monocytes from lymphocyte populations. An average of 9.45% versus 1.22% of cells, within chosen lymphocyte gates established by forward angle and 90 degrees scatter, respectively, were identified as monocytes. In samples from ten donors, the exclusion of monocytes from the lymphocyte population was more efficient using 90 degrees scatter than forward scatter. Simultaneous use of forward and 90 degrees scatter did not significantly improve the ability to accurately exclude monocytes, but did result in a significant increase in the improper exclusion of lymphocytes. Use of 90 degrees scatter alone, forward scatter alone, and forward and 90 degrees scatter simultaneously to identify lymphoid cells resulted in the exclusion of 12, 17, and 23% of lymphocytes from further analysis. The 90 degrees scatter alone appears to be the optimal method to eliminate monocytes electronically from mononuclear cell populations in which lymphocytes are being studied.     

Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.     

Phorbol-induced recruitment of lymphocytes for spontaneous cytolysis of natural killer-insensitive tumor targets

Natural killer (NK) cells lyse a variety of tumor cells in vitro whereas NK-depleted unsensitized lymphocytes do not have this effect. In studies designed to elucidate the NK phenomenon, a series of experiments was carried out to identify properties of NK-sensitive targets and compare these with those of NK-insensitive targets and with targets rendered sensitive by treatment with phorbol esters. Following brief exposure to phorbol-12-myristate-13-acetate (PMA), the targets were thoroughly washed, and then incubated with lymphocyte preparations which were either enriched for or depleted of NK cells. PMA treatment increased the susceptibility of sensitive targets to NK-enriched fractions by only 20-30%, but made the NK-cell-insensitive targets markedly vulnerable to these effectors (80% lysis). Unexpectedly, brief PMA exposure also rendered cells susceptible to lysis by NK-cell-depleted lymphocytes. Yet, such targets were not killed by monocytes or B lymphocytes. Elimination of T8 lymphocytes from the NK-depleted fractions abolished lysis. To explore whether PMA had induced membrane changes not detectable on electron microscopy of thin sections, freeze-fracture studies were carried out on target cells before and after treatment with PMA. Freeze-fracture replicas of target cells which had been exposed to PMA exhibited a 50% reduction of the intramembranous particles (IMP) on the external leaflet of the plasma membrane but no changes in the number or size of the IMP associated with the protoplasmic leaflet face. The exact relationship of the structural changes and enhanced susceptibility to cytolysis has not yet been established. However, the observation that normal and tumor cells can be rendered vulnerable to lysis by lymphocytes which have not been sensitized immunologically may have practical applications.     

In search of alpha 1-microglobulin on the lymphocyte surface.

alpha 1-Microglobulin was found by immunofluorescence not to be associated with human lymphoid and nonlymphoid cell lines. No accumulation of alpha 1-microglobulin was detected in culture media of these cell lines. A weak membrane fluorescence with anti-alpha 1-microglobulin on peripheral lymphocytes could not be blocked by the purified protein. No release of alpha 1-microglobulin into the growth medium was seen by normal cultured leukocytes. Treatment of normal lymphocytes, erythrocytes, and various cell lines with solubilization techniques did not yield any alpha 1-microglobulin. alpha 1-Microglobulin and protein HC display immunologic and biochemical identity. However, anti-protein HC stained almost all of the tested cell lines and normal lymphocytes. Blocking experiments with the purified protein were not successful. Antibodies reacting with a minor impurity (50,000 d) in the alpha 1-microglobulin or protein HC preparations could be absorbed from anti-alpha 1-microglobulin with normal leukocytes and a lymphoid cell line.     

SC3 monoclonal antibody defines a novel specific human B-cell surface antigen differentially expressed on B-cell leukaemias and lymphomas and involved in the proliferation of normal and malignant B lymphocytes

The monoclonal antibody SC3 was raised against the NK leukaemia cell line YTindi. It detected a 98-kDa surface antigen with weak expression on a restricted number of leukaemia cell lines under reducing conditions. SC3 mAb labelled 5-10% of normal peripheral blood lymphocytes corresponding almost exclusively to B lymphocytes, and 60-70% of tonsillar B cells. It did not react with erythrocytes, platelets or monocytes whereas it stained granulocytes. The aim of the present study was to examine the expression and functional effects of SC3 mAb reactive epitope on normal and malignant B cells. Most SC3+ B cells from healthy donors were CD23+, some co-expressed CD5 and CD27 and a few were CD38+. SC3 epitope was expressed exclusively by B-lineage malignant proliferations, including B-lineage ALL. Practically, all B-CLL studied expressed SC3 mAb reactive epitope although with variable intensity, while MCL and PLL were negative. Other low grade and high grade B-NHL were variably stained. SC3 mAb alone triggered the proliferation of CD2-depleted PBL and significantly increased the proliferation induced by suboptimal concentrations of LPS. This effect was much weaker with B-CLL cells but was increased after cross-linking with an anti-IgM antibody. The restricted expression pattern combined with molecular weight and functional data indicate that SC3 mAb may detect a novel B-cell antigen mostly expressed by early and naive B cells. Although its expression in B-cell malignancies was not limited to a single differentiation stage, it might confer specific functional characteristics to the positive malignant cells.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Antigen presentation by human monocytes: evidence for stimulant processing and requirement for interleukin 1

We studied the role of stimulant processing and presentation and of IL 1 in monocyte-mediated activation of human lymphoproliferative responses. The effects of two lysosomotropic agents, ammonium chloride and chloroquine, on the capacity of human monocytes to activate T lymphocyte responses to the soluble antigen streptolysin O (SLO) and to the polyclonal stimulant S. aureus protein A (SpA) were investigated. These agents inhibited the presentation of SLO and SpA by human monocytes in a dose-dependent manner. The inhibition occurred if monocytes were treated with ammonium chloride and chloroquine for 1.5 hr, starting only 30 min after exposure to the stimulants, whereas only minimal inhibition occurred when monocytes were treated with the two lysosomotropic compounds 2 hr after pulsing with SLO or SpA. In contrast, cell membrane alloantigen presentation by monocytes in the MLR was not affected by ammonium chloride or chloroquine treatment. Thus, these reversible inhibitors of monocyte phagosome-lysosome functions presumably interfere with intracellular processing of the stimulants but do not seem to interfere with alloantigen presentation at the cell surface. Furthermore, we investigated whether gently fixed monocytes were still capable of passively presenting stimulant or whether active metabolic processes as well as IL 1 were required. We observed that only monocytes treated with paraformaldehyde after SLO or SpA pulsing stimulated a proliferative response by T lymphocytes, provided 50 U/ml of partially purified human IL 1 were added back to cultures. In contrast, monocytes fixed before exposure to SLO or SpA were not able to stimulate T lymphocytes even if supplemented by IL 1. Taken together these data suggest that a finite incubation period is required for human monocytes to become able to present SLO or SpA to T lymphocytes. During this time the soluble stimulants presumably undergo some metabolic process in viable macrophages perhaps at the phagosome-lysosome level, to become recognizable by T lymphocytes.     

Effects of blood monocytes and lymphocytes on progesterone secretion by granulosa cells in the pig

We studied the effects of autologous and nonautologous co-cultures of porcine blood monocytes and lymphocytes with granulosa cells on progesterone secretion. Eight prepubertal crossbred gilts were ovariectomized, and the granulosa cells were collected, plated at 2.5 x 10(5) cells/ml and allowed to attach. Blood was obtained from the same eight gilts, and the mononuclear cells were separated by density gradient centrifugation. Monocytes were separated from lymphocytes by adherence to plastic. Adherent monocytes, lymphocytes and a 1:1 mixture of monocytes + lymphocytes were added to granulosa cell cultures and incubated for 48 h. Progesterone secretion into the media was measured. In addition, blood cell alloreactivity was studied in these co-cultures by measuring uptake of (3)H-thymidine. The co-culture of adherent monocytes or monocytes + lymphocytes with granulosa cells increased (P <.05) progesterone secretion as compared with granulosa cells cultured alone. However, co-culture of lymphocytes with granulosa cells did not have a significant effect. No difference was observed between autologous and nonautologous cell cultures in blood cell proliferation or granulosa cell progesterone secretion. In conclusion, blood monocytes influence progesterone secretion by granulosa cells. In addition, there was no difference in the ability of autologous and nonautologous blood cells to stimulate progesterone secretion by granulosa cells. No alloreactivity was observed using nonautologous immune cells with granulosa cells.     

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes.

Human blood T-lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol.1 cell water-1.h-1) was half that in normal blood T-lymphocytes (20 mmol.1 cell water-1 h-1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K+ transport was 14 mmol.1 cell water-1.h-1 and treatment with PHA increased K+ transport only 30%. The intermediate values of basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data suggest that B-lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.     

Suppression of a pokeweed mitogen-stimulated plaque-forming cell response by a human B lymphocyte-derived aggregated IgG-stimulated suppressor factor: suppressive B cell factor (SBF)

The mechanisms whereby formed immune complexes (IC) or immunoglobulin aggregates can suppress further antibody production were explored by culturing normal human peripheral blood mononuclear leukocytes (PBL) with heat-aggregated IgG (HAIgG) and collecting the culture supernatants at 24 hr. These supernatants were found to suppress a pokeweed mitogen (PWM)-induced rheumatoid factor plaque-forming cell (RF-PFC) response in normal individuals. PWM-induced anti-trinitrophenylated sheep red blood cell (TNP-SRBC) PFC were also inhibited by suppressor supernatants from HAIgG-stimulated PBL, suggesting that the polyclonal PFC response was inhibited by a suppressor factor. The suppressor factor inhibited PWM stimulated RF-PFC throughout the culture period, but suppression was maximal at the peak of the RF-PFC response. Suppressor factor was only effective at the initiation of cultures, suggesting that it inhibited early events in the PWM-stimulated RF-PFC response. Molecular weight determination of the suppressor factor by differential membrane fractionation suggested a m.w. range of 30,000 to 50,000, and chromatography on Sephadex G-100 showed a peak activity at an approximate m.w. of 32,000. Studies suggested the factor was not an interferon. Depletion of T lymphocytes by E rosetting and macrophages/monocytes by G-10 adherence did not affect the generation of suppressor factor. Depletion of T lymphocytes (OKT4, OKT8) and NK cells (Leu-11b) by antibody-dependent, complement-mediated cytotoxicity also did not affect the generation of suppressor factor. Depletion of B lymphocytes with OKB7 resulted in the generation of significantly less suppressor factor. Suppression produced by unstimulated purified B lymphocytes was approximately one-half that seen when B lymphocytes were stimulated with HAIgG. Differential membrane fractionation studies suggested that only HAIgG-stimulated B cell cultures contained peak activity in the 30,000 to 50,000 m.w. fraction. Supernatants from unstimulated purified T cells also generated suppression, which was approximately one-half of that seen with HAIgG-stimulated B cells, but no increase in suppressor activity was seen in T cell cultures after incubation with HAIgG. These studies demonstrate that HAIgG is capable of stimulating B lymphocytes to produce a lymphokine, suppressive B cell factor (SBF), which is capable of suppressing a polyclonal PFC response. SBF may be important in feedback control of human immunoglobulin production.     

The evolution of MHC restrictions in antigen recognition by T cells in a haploidentical bone marrow transplant recipient

We have longitudinally followed the major histocompatibility complex (MHC) restrictions that govern the response of T lymphocytes to specific Ag in a child with severe combined immunodeficiency who was successfully transplanted by using T cell depleted haploidentical maternal bone marrow cells and immunized shortly afterwards with tetanus toxoid (TT) Ag. In the first year post-transplant, monocytes were of both donor and recipient origin whereas T and B cells were of donor origin. Three years after transplant, all monocytes and T and B cells were of donor origin. T lymphocytes taken from the child at that time and depleted in vitro of alloreactivity to paternal Ag proliferated in response to TT presented by maternal as well as paternal monocytes. A TT-specific T cell line established from these cells in the presence of maternal monocytes cooperated with maternal but not with paternal monocytes, whereas a TT-specific T cell line established in the presence of paternal monocytes cooperated with paternal but not with maternal monocytes and with monocytes derived from a paternal uncle who shared the haplotype inherited by the recipient from her father. These results show that long-term memory T cells restricted to recipient MHC Ag not shared with the bone marrow donor continue to circulate long after the disappearance of accessory cells of recipient origin. These T cells could potentially participate in a secondary immune response because they were shown to recognize TT presented by recipient fibroblasts induced to express class II MHC molecules following treatment with IFN-gamma.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.     

Establishment of a human T cell hybridoma cell line producing suppressor factor specific for anti-thyroglobulin antibody production

Thyroglobulin (Tg)-binding peripheral blood T cells from a normal individual were fused with a T cell leukemia cell line (Jurkat-AG9) treated by emetine and actinomycin D. Several cell lines were established from thus-prepared human T cell hybridomas. The culture supernatant from one of these lines (Tg-Ts47) whose phenotype was OKT3- 11+ 4+ 8- suppressed the generation of Tg-specific antibody-forming cells from the lymphocytes of patients with Hashimotos' chronic thyroiditis, but not anti-SRBC and anti-ovalbumin antibody production from both autologous and patient lymphocytes. Tg-Ts47-derived factors also bore Tg antigen-binding sites. The suppressive activity of the supernatants was shown in almost all patients lymphocytes tested. This indicated that the supernatants of Tg-Ts47 line contain a suppressive factor specific for Tg antigen and capable of acting across allogeneic barriers.     

Ultrastructural patterns of the alpha-naphthyl butyrate esterase activity in normal and leukaemic peripheral blood leucocytes

Summary The activity of -naphthyl butyrate esterase was investigated at the ultrastructural level in normal human peripheral blood and in a few cases of hairy cell leukaemia, B-chronic lymphocytic leukaemia and acute monocytic leukaemia. A membrane reactivity was detected in most normal monocytes and lymphocytes. The activity in monocytes was very strong and was inhibited by NaF. It was NaF-resistant and less intense in lymphocytes. The reaction product was localized in the cytoplasm only in a small percentage of lymphocytes.In lymphocytes and monoblasts from pathological samples the pattern of reactivity was similar to that found in their normal counterparts, except for a lower intensity. The hairy cells showed a discrete distribution of the NaF-resistant reaction product on their cell surface.The different patterns of enzyme distribution are discussed critically.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

Control of aromatase activity in breast tumours: the role of the immune system

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.     

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.     

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.     

Role of class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes

The capacity of peripheral blood monocytes and B lymphocytes to support staphylococcal protein A (SpA)-induced proliferation of autologous and allogeneic T cells, as well as the role of major histocompatibility complex (MHC) class I and II molecules in this activation process, were investigated. Highly purified peripheral T lymphocytes did not proliferate in response to SpA, but their response was reconstituted by both irradiated (or mitomycin C-treated) monocytes and B lymphocytes. The effect of B cells on the SpA-induced T-cell response could not be explained by a contamination of residual accessory cells because long-term continuous B-cell lines restored SpA-induced T-cell DNA synthesis as effectively as did monocytes. Support of SpA responsiveness by B cells could not be accounted for by polyclonal binding of SpA to cell surface immunoglobulins, since the ability of SpA-unreactive and SpA-reactive B cells was comparable. The cells from two human leukemic lines--K562 and Raji--showed the same ability in supporting the pokeweed mitogen-induced T-cell response, but the class II-positive Raji cells were much more effective than class II-negative K562 cells in restoring the T-cell responsiveness to SpA. Monoclonal antibodies specific for monomorphic determinants of MHC class II antigens, as well as their F(ab')2 fragments, consistently inhibited the SpA-induced proliferative response, whereas antibodies specific for MHC class I antigens were without effect. The antibodies specific for class II antigens appeared to act at the level of accessory cell, since pretreatment with these antibodies inhibited the ability of SpA-pulsed monocytes or Raji cells to present SpA to autologous or allogeneic T lymphocytes, respectively. These data indicate that either monocytes or normal and lymphoblastoid B cells can act as accessory cells for the proliferative response of human T cells to soluble SpA and that monomorphic determinants of MHC class II molecules play an important role in this activation process.     

Mechanism of suppression of human peripheral blood lymphocyte proliferation by platelet activation factor

The influence of the phospholipid platelet activation factor (PAF) was studied on PHA-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that influencing on the whole population of lymphocytes PAF suppressed proliferation mainly of T-cells. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the suppression of lymphocyte proliferation induced by PHA. Pretreatment of lymphocytes with PAF antagonist--BL 8705 almost completely blocked the suppression of lymphocyte proliferation induced by PHA.