Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.     

Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.     

Suppression of mitogen-induced lymphocyte proliferation by syringomycin-E

Abstract Syringomycin E (SR-E) is low molecular weight bacterial lipodepsipeptide with antifungal properties. Owing to immunosuppressive activities of such compounds as cyclosporine, FK506 and rapamycin, we studied the effect of SR-E on proliferation of human blood lymphocytes in vitro. SR-E, by itself, had no effect but the mitogen-induced lymphocyte proliferation was significantly suppressed. The suppressive effect was more pronounced with pokeweed mitogen (PWM) as compared to phytohemagglutinin (PHA) or monoclonal antibody to CD3 (anti-CD3). Since these mitogens induce cellular immunity (T cell-dependent), SR-E may potentially be a novel immunosuppressive compound.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Suppression of lymphocyte reactivity by culture supernatant from horse embryos and endometrium

The mechanisms that permit maternal tolerance of the conceptus allograft during early pregnancy in the mare have not been investigated. Embryos and endometria were collected from mares 14 days after ovulation and cultured for 20.5 h. The effect of addition of culture supernatant on incorporation of [3H]thymidine by equine peripheral blood lymphocytes was studied. Culture supernatant from endometrium of nonpregnant mares did not affect lymphocyte blastogenesis, but supernatant from both embryos and endometrium of pregnant mares reduced concanavalin A (Con A)- and phytohemagglutinin-induced blastogenesis. Five of six cultures performed in the present of indomethacin did not contain immunosuppressive factors. The suppressive effect on Con A-induced blastogenesis was eliminated by charcoal treatment of the supernatants and reduced by treatment with trypsin or heat. Blastogenesis of bovine lymphocytes was inhibited by culture supernatant of endometrium from pregnant mares, but not by embryo supernatant. Preincubation of blood lymphocytes with supernatants from endometrium of pregnant mares enhanced subsequent incorporation of [3H]thymidine by lymphocytes. A 24-h delay in addition of embryo culture supernatants significantly reduced the degree of immunosuppression. These results suggest that probably more than one substance interacts with the lymphocyte cultures and the observed blastogenesis reflects the end result of the interaction between suppressive and stimulating factors. The lymphocyte inhibitory effect evident in supernatants from embryos and endometrium from pregnant mares may be important in local immunosuppression and maternal acceptance of pregnancy.     

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 effects of crude and purified human gonadotropin on in vitro stimulated human lymphocyte cultures.

Crude human chorionic gonadotropin (hCG) was found to be several fold more immunosuppressive than purified hCG in human peripheral blood lymphocyte cultures stimulated by phytohemagglutinin, pokeweed, purified protein derivative and allogeneic cells in vitro. Immunosuppression by crude hCG was consistently noted at levels less than 1000 IU/ml and usually 80% inhibition was achieved with doses of 5000–10,000 IU/ml, whereas 40–50% inhibition or less was observed by purified hCG at 10,000 IU/ml. In two crude hCG preparations subjected to Sephadex G-100 chromatography, the fractions that inhibited lymphocyte cultures appeared in the eluate after the major peak of hCG activity. These data indicate that inhibitory substance(s) other than hCG are responsible for most of the immunosuppressive properties of first trimester pregnancy urine. Both crude and purified hCG were stimulatory to human lymphocytes when used alone without mitogens when cultured in fetal calf serum.     

Inhibition of interleukin 1 activity by a factor in submandibular glands of rats

With the sequential use of ammonium sulfate precipitation, gel filtration and chromatofocusing, we have partially purified from extracts of the submandibular glands of rats a factor (referred to as submandibular gland's immunosuppressive factor or SMG-ISF) capable of inhibiting the in vitro proliferation of mitogen- and antigen-stimulated murine lymphocytes. The semi-purified suppressor fractions had an isoelectric point of 4.4 to 4.5 and consisted of at least three molecular species. These active fractions suppressed the mitogenic effects of Concanavalin A phytohemagglutinin, and lipopolysaccharide. In vitro immune reactions such as the mixed lymphocyte culture MLC reaction and the production of cytotoxic T lymphocytes (CTL) across major histocompatibility barriers in mice were also suppressed. These in vitro immunosuppressive effects required the addition of the suppressor fractions early after the initiation of the cultures and were reversed if the factor was removed from the cultures at least 48 to 72 hr before the completion of the assays. The active fractions did not affect the proliferation of CTLL 2 cells induced by interleukin 2 (IL 2), but inhibited the mitogenic and co-stimulatory effects of IL 1 on mouse thymocytes, and in this effect showed a dose-response relation suggestive of a competitive mechanism. These characteristics of SMG-ISF indicate a specific inhibition of the activity of IL 1.     

A mutant human T-cell line producing immunosuppressive factor(s)

6T-CEM-20, a subclone of a 6-thioguanine-resistant mutant derived from the human-T-cell line CEM, secreted into the medium, a high titered immunosuppressive factor specific for T cells. The cell-free supernatant was very potent in suppressing, via a noncytotoxic mechanism, mitogen-activated T-cell proliferation, cytotoxic T-cell functions, and pokeweed mitogen (PWM)-stimulated plaque-forming cells (PFC). Lower dilutions of the supernatant (10(-1)-10(-2] abrogated T-cell functions within 24 hr whereas higher dilutions (10(-3)-10(-7] required a culture period of up to 4 days with lymphocytes to arrest T-cell activities. The suppressive activity was most pronounced when the factor was added in the early part of the culture period. The factor was sensitive to heat treatment and both low and high pH (most stable at physiological pH). Preliminary purification with column chromatography indicates that the active moiety was contained in the high-molecular-weight fraction (MW greater than 200,000). Data from coculture experiments suggested that T lymphocytes, which were exposed for 5-12 hr to the active supernatant or the partially purified material, suppressed allogeneic T-cell proliferation in a dose-related manner. This suppressor factor which we called suppressor-activating factor (SAF) might have activated a suppressor population or induced the production of a suppressor factor which in turn mediated the observed suppression. Both the molecular structure and the detailed mechanism of action are under investigation.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

Proliferation of PHA- and PWM-stimulated lymphocytes measured by sister chromatid differential staining.

Lymphocyte proliferation in PHA- and PWM-stimulated cultures was measured by BrdU incorporation followed by sister chromatid differential staining. Both the PHA and PWM cultures showed a steady increase with time in the number of second- and third-division metaphases, accompanied by a corresponding decrease in the number of first-division metaphases. There was no difference between the PHA and PWM cultures in the number of first-, second-, and third-division cells, but the mitotic index was lower in the PWM cultures. During the 96-hr culture period the mitotic index of the PHA cultures decreased, whereas the PWM cultures showed an increase. It is suggested that the BrdU-Giemsa technique may prove to be a useful adjunct in the study of lymphocyte proliferation in response to mitogens.     

Immunophenotype and sister chromatid differentiation: a combined methodology for analyzing cell proliferation in unfractionated lymphocyte cultures

Combination of the MAC (morphology, antibody, chromosomes) and harlequin staining procedures offers a method for direct analysis of cell kinetics in cultures of unfractionated hematopoietic cells. In the present study unfractionated human mononuclear leukocyte cultures were stimulated with PHA or PWM mitogens and exposed to bromodeoxyuridine for various periods. For MAC, cytospin preparations were made and cells were classified with monoclonal B and T antibodies by the immunoperoxidase technique. After differentiation of the different lymphocyte subsets, the cells were stained by a fluorescence-plus-Giemsa method to distinguish sister chromatids and to determine the proportions of first, second, third, or subsequent mitoses among the previously identified subsets. The results showed (1) that the relative proportions of mitotic T and B cells are the same regardless of the mitogen used; (2) that T and B lymphocytes proliferate faster in cultures stimulated by PWM than in those stimulated by PHA; and (3) that T cells enter mitosis earlier than B cells when PHA or PWM are used as mitogens.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

Immunomodulatory effects of fusarochromanones TDP-1 and TDP-2.

An in vitro peripheral lymphocyte blastogenesis system was used to investigate the biological activities of the fungal toxin fusarochromanone (TDP-1) and its monoacetyl derivative TDP-2. Briefly, cultures of human or bovine peripheral lymphocytes were exposed to TDP-1 or TDP-2 and a mitogen (PHA, Con A or PWM). After a standard incubation time, cell proliferation was quantified using the MTT bioassay. Human and bovine lymphocyte proliferation was inhibited by high concentrations of TDP-1; however, bovine lymphocyte proliferation was significantly increased at low concentrations of TDP-1. TDP-2 has similar but less pronounced effects on lymphocyte proliferation.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Human B cell differentiation. II. Suppression by T cells of T-dependent and T-independent plasma cell maturation.

In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.     

Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

Lipoproteins may provide fatty acids necessary for human lymphocyte proliferation by both low density lipoprotein receptor-dependent and -independent mechanisms

Human lymphocytes respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with transferrin if fatty acids necessary for maximal proliferation are provided. Either lipoproteins or exogenous fatty acids support optimal lymphocyte responses. The current studies examined the role of cell surface receptors for low density lipoprotein (LDL) in the enhancement of lymphocyte proliferation. Support of lymphocyte growth by limiting concentrations of LDL was found to involve interaction of the lipoprotein with LDL receptors. Thus, modification of LDL by reductive methylation so as to inhibit receptor-mediated interactions markedly decreased the capacity of LDL to enhance lymphocyte proliferation. Moreover, growth of lymphocytes obtained from patients with LDL receptor-negative homozygous familial hypercholesterolemia was minimal when cultures were supplemented with low concentrations of LDL (less than 10 micrograms cholesterol/ml). LDL also enhanced lymphocyte proliferation by a receptor-independent mechanism since high concentrations (greater than or equal to 50 micrograms cholesterol/ml) supported growth of both normal and familial hypercholesterolemia lymphocytes. In contrast, support of lymphocyte proliferation by high density lipoprotein (HDL) subclass 3 was completely independent of LDL receptors. Thus, HDL3 enhanced responses of both normal and familial hypercholesterolemia lymphocytes in an equivalent concentration-dependent manner; this effect was not altered by reductive methylation of HDL3. One function of lipoproteins in this system may be the provision of fatty acids since oleic and linoleic acids enhanced DNA synthesis by both normal and familial hypercholesterolemia lymphocytes in the absence of lipoproteins. These results indicate that lipoproteins may provide fatty acids necessary for optimal proliferation of human lymphocytes by both LDL receptor-mediated and LDL receptor-independent interactions.     

Heat stress-induced alterations in the synthesis and secretion of proteins and prostaglandins by cultured bovine conceptuses and uterine endometrium

Effect of in vitro heat stress on protein and prostaglandin synthesis and secretion by bovine conceptuses and endometrium was examined. Conceptuses (n = 11) and endometrium (n = 10) obtained on Day 17 of pregnancy were cultured at thermoneutral (39 degrees C, 24 h) or heat stress (39 degrees C, 6 h; 43 degrees C, 18 h) temperatures in medium supplemented with L-[4,5-3H]leucine (100 microCi) and arachidonic acid (10 micrograms/ml). Radiolabeled protein secreted into culture medium increased with time in both groups. Heat stress reduced (p less than 0.001) incorporation of [3H]leucine into intracellular and secreted proteins by conceptuses but did not alter incorporation of [3H]leucine by endometrium. In particular, heat stress reduced by 72% the secretion of bovine trophoblast protein-1, the conceptus polypeptide believed to cause extension of luteal lifespan. Two-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that heat stress altered the array of proteins in endometrial and conceptus tissues, as evidenced by the induction of "heat-shock proteins." Endometrial secretion of prostaglandin F (p less than 0.001) and conceptus secretion of prostaglandin E2 (p less than 0.05) increased in response to heat stress. Sensitivity of bovine conceptuses and endometrium to heat stress in vitro suggests that infertility associated with maternal heat stress may be caused, partially by alterations in signals required for maintenance of the corpus luteum during early pregnancy.