Arzoxifene, a new selective estrogen receptor modulator for chemoprevention of experimental breast cancer.

Arzoxifene ([6-hydroxy-3-[4-[2-(1-piperidinyl)-ethoxy]phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiophene) is a selective estrogen receptor modulator (SERM) that is a potent estrogen antagonist in mammary and uterine tissue while acting as an estrogen agonist to maintain bone density and lower serum cholesterol. Arzoxifene is a highly effective agent for prevention of mammary cancer induced in the rat by the carcinogen nitrosomethylurea and is significantly more potent than raloxifene in this regard. Arzoxifene is devoid of the uterotrophic effects of tamoxifen, suggesting that, in contrast to tamoxifen, it is unlikely that the clinical use of arzoxifene will increase the risk of developing endometrial carcinoma.

Photochemical synthesis of N-arylbenzophenanthridine selective estrogen receptor modulators (serms).

Selective estrogen receptor modulators are an emerging class of pharmaceutically important molecules. Many compounds in this class contain an aminoethoxyaryl moiety attached to a polycyclic framework at an asymmetric carbon atom. To assess whether this carbon atom can be replaced by nitrogen, we have employed a Ninomiya enamide photocyclization for the rapid synthesis of a novel N-arylbenzophenanthridine framework, 4. Further elaboration of 4 into a new structural class of achiral, nonsteroidal estrogen receptor modulators is described.

An estrogen receptor basis for raloxifene action in bone.

Although controversy remains regarding direct effects of estrogen on bone, in vivo data clearly show that estrogens suppress bone turnover, resulting in decreased bone resorption and formation activity. Selective estrogen receptor modulators (SERMs), such as raloxifene, produce effects on bone which are very similar to those of estrogen. In vitro, both raloxifene and estrogen inhibit mammalian osteoclast differentiation and bone resorption activity, but only in the presence of IL-6. Data from a number of ovariectomized rat model manipulations (i.e. hypophysectomy, low calcium diet and drug combinations) demonstrate a strong parallel between the antiosteopenic effects of raloxifene and estrogen. A characteristic action of estrogens on the skeleton is inhibition of longitudinal bone growth, an effect which is not observed with other resorption inhibitors, including calcitonin and bisphosphonates. Consistent with an estrogen-like mechanism on bone, raloxifene inhibits longitudinal bone growth in growing rats. In addition to the overall similarity of the bone activity profile in animals, estrogen and raloxifene also produce similar effects on various signaling pathways relative to the antiosteopenic effect of these two agents. For example, IL-6, a cytokine involved in high turnover bone resorption following estrogen deficiency in rats, is suppressed by both raloxifene and estrogen. Raloxifene and estrogen also produce a similar activation of TGF-beta3 (a cytokine associated with inhibition of osteoclast differentiation and activity) in ovariectomized rats. Like 17beta-estradiol, raloxifene binds with high affinity to both estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta). Crystal structure analyses have shown that 17beta-estradiol and raloxifene bind to ER alpha with small, but important, differences in three dimensional structure. These subtle differences in the conformation of the ligand:receptor complex are likely the basis for the key pharmacological differences between estrogens and the various SERMs (i.e. raloxifene vs tamoxifen). Raloxifene also produces estrogen-like effects on serum cholesterol metabolism and the vasculature. Thus, while raloxifene exhibits a complete estrogen antagonist in mammary tissue and the uterus, it produces beneficial effects on the cardiovascular system and prevents bone loss via an estrogen receptor mediated mechanism.

Synthesis and biological activity of trans-2,3-dihydroraloxifene.

The synthesis and biological evaluation of trans-2,3-dihydroraloxifene, 2, is described. The synthesis proceeds in 8 steps in 20% overall yield. Relative trans 2,3-stereochemistry is definitively established in ester 6, which is converted to the title compound via derivatization, Grignard addition, and deprotection. Evaluation in vitro shows the compound to be a potent selective estrogen receptor modulator (SERM).

Synthesis and pharmacology of conformationally restricted raloxifene analogues: highly potent selective estrogen receptor modulators.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.

Discovery and synthesis of [6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-hydroxyphenyl)]b enzo[b]thiophene: a novel, highly potent, selective estrogen receptor modulator.

Raloxifene,[2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl] [4-[2-(1-piperidinyl)ethoxy]phenyl]methanone hydrochloride (2), is representative of a class of compounds known as selective estrogen receptor modulators (SERMs) that possess estrogen agonist-like actions on bone tissues and serum lipids while displaying potent estrogen antagonist properties in the breast and uterus. As part of ongoing SAR studies with raloxifene, we found that replacement of the carbonyl group with oxygen ([6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-hydroxyphenyl)]b enzo[b]thiophene hydrochloride, 4c) resulted in a substantial (10-fold) increase in estrogen antagonist potency relative to raloxifene in an in vitro estrogen dependent cell proliferation assay (IC50 = 0.05 nM) in which human breast cancer cells (MCF-7) were utilized. In vivo, 4c potently inhibited the uterine proliferative response to exogenous estrogen in immature rats following both sc and oral dosing (ED50 of 0.006 and 0.25 mg/kg, respectively). In ovariectomized aged rats, 4c produced a significant maximal decrease (45%) in total cholesterol at 1.0 mg/kg (p.o.) and showed a protective effect on bone relative to controls with maximal efficacy at 1.0 mg/kg (p.o.). These data identify 4c as a novel SERM with greater potency to antagonize estrogen in uterine tissue and in human mammary cancer cells compared to raloxifene, tamoxifen or ICI-182,780.

Evaluation of the major metabolites of raloxifene as modulators of tissue selectivity.

Raloxifene (LY139481 HCl) is a selective estrogen receptor modulator (SERM) which blocks the effects of estrogen on some tissues, such as the breast and uterus, while mimicking estrogen in other tissues, such as bone. To study the origins of this unique pharmacology, we have prepared the major metabolites of raloxifene as chemical probes for examining the estrogen receptor function in vitro and in vivo. In human breast cancer cell (MCF-7) related assays, these glucuronide conjugates show little affinity for the estrogen receptor and are more than two orders of magnitude less potent at inhibiting cell proliferation than raloxifene. In non-traditional estrogen target tissue, such as bone, these metabolites are less effective than the parent at inhibiting cytokine-stimulated bone resorbing activity in rat osteoclasts or producing transforming growth factor beta-3 (TGF-beta3). In animal models, tissue distribution studies with radiolabelled metabolite indicate that conversion to raloxifene occurs readily in a variety of tissues including the liver, lung, spleen, kidney, bone and uterus. Differential conversion of metabolite in target organs, such as bone and the uterus, is not observed indicating that the origin of raloxifene's pharmacology does not result from tissue-selective deconjugation of metabolite to parent.

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.

Hypocholesterolemic activity of raloxifene (LY139481): pharmacological characterization as a selective estrogen receptor modulator.

After once-daily oral dosing in ovariectomized rats, raloxifene (LY139481) hydrochloride produced dose- and time-dependent reductions in serum cholesterol and high-density lipoprotein-cholesterol. Paired-feeding studies demonstrated that effects of raloxifene on serum lipids were not secondary to effects on food consumption. Maximal reductions in serum cholesterol occurred within 4 days of raloxifene administration or sooner, depending on the administered dose. The ED50 for 50% reduction in serum cholesterol by raloxifene was 0.13 +/- 0.04 mg/kg/day (mean +/- S.E.M., n = 17); maximal cholesterol reduction by raloxifene (68%) was significantly less than that produced by estrogen (17 alpha-ethinylestradiol; 89%) after 4 to 7 days of daily dosing. Dose-response curves for cholesterol lowering by raloxifene were generated in the presence of varying doses of 17 alpha-ethinylestradiol; two-way analysis of variance revealed significant interactions between estrogen and raloxifene with respect to cholesterol lowering (P < .001). Furthermore, a high dose of raloxifene (10 mg/kg/day) prevented further reduction of serum cholesterol by estrogen (1-100 micrograms/kg/ day) beyond that produced by raloxifene alone. For a series of closely related structural analogs of raloxifene, log(ED50) values for cholesterol lowering were highly correlated with log(relative binding affinity) for the estrogen receptor (r = 0.93; P < .0001). Thus, cholesterol lowering by raloxifene in ovariectomized rats is mediated primarily via partial agonist effects at estrogen receptors. Taken together with previous observations in uterine tissue of estrogen antagonism by raloxifene in the absence of significant agonism, the present findings support the classification of raloxifene as a selective estrogen receptor modulator.

Molecular determinants of tissue selectivity in estrogen receptor modulators.

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (A) the presence of phenolic hydroxyls, (B) different substituents on the basic amine, (C) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (D) the imposition of a carbonyl "hinge" between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.

Estriol: a potent regulator of TNF and IL-6 expression in a murine model of endotoxemia.

The increased incidence of autoimmune disease in premenopausal women suggests the involvement of sex steroids in the pathogenesis of these disease processes. The effects of estrogen on autoimmunity and inflammation may involve changes in the secretion of inflammatory mediators by mononuclear phagocytes. Estradiol, for example, has been reported to regulate TNF, IL-6, IL-1 and JE expression. In the present study the effects of the estrogen agonist, estriol, on cytokine expression have been investigated in mice administered a sublethal lipopolysaccharide, LPS, challenge. Pretreatment of mice with pharmacologic doses of estriol, 0.4-2 mg/kg, resulted in a significant increase in serum TNF levels in both control and autoimmune MRL/lpr mice, following LPS challenge. This increase in TNF over the placebo group was blocked by the estrogen antagonist tamoxifen. Estriol treated mice also exhibited a rapid elevation in serum IL-6 levels following LPS challenge with the peak increase occurring 1 hr post LPS. This contrasted with the placebo group in which maximal serum IL-6 levels were detected at 3 hrs post challenge. This shift in the kinetics of IL-6 increase by estriol was inhibited by tamoxifen. The estriol mediated effects of TNF and IL-6 serum levels were consistent with the changes in TNF and IL-6 mRNA observed ex vivo in elicited peritoneal macrophages. Macrophage cultures from estriol treated animals however, did not demonstrate significant differences from the placebo group for TNF or NO secretion following in vitro LPS challenge. These results suggest that the estrogen agonist estriol can have significant quantitative, TNF, and kinetic, IL-6, effects on inflammatory monokines produced in response to an endotoxin challenge.

Environmental estrogens: effects on cholesterol lowering and bone in the ovariectomized rat.

Representative non-steroidal estrogens, from common environmental sources such as plants, pesticides, surfactants, plastics, and animal health products, demonstrated an ability to lower serum cholesterol and prevent bone loss. Specifically, select environmental estrogens (coumestrol, genistein, methoxychlor, bisphenol A, and zeranol) effectively lowered total serum cholesterol in an estrogen-dependent animal model, the ovariectomized rat. Of these entities, coumestrol, methoxychlor, and zeranol prevented ovariectomy-induced bone loss. In an in vitro environment, these compounds competed with 17beta-estradiol for estrogen receptor binding and stimulated cell proliferation in a human breast cancer cell line (MCF-7). In addition to their well-documented effects on reproductive tissue, various environmental estrogens can dramatically affect non-reproductive parameters such as cholesterol lowering and bone metabolism.

A role for CD8+ T lymphocytes in osteoclast differentiation in vitro.

Bone loss associated with chronic inflammatory diseases has been attributed to the release of cytokines from T lymphocytes. However, the role of T lymphocyte subsets in the mediation of osteoclast activity has not been extensively studied. Cocultures of murine bone marrow and BALC cells (murine calvarial-derived cell line) were used to study osteoclast differentiation. Murine marrow was left intact or depleted of cells expressing the CD8 or CD4 antigen by immunomagnetic separation and then cocultured with BALC cells in the presence or absence of 1,25-(OH)2D3. Depleting bone marrow of CD4-positive (CD4+) cells did not affect osteoclast differentiation (formation of tartrate-resistant acid phosphatase positive cells with three or more nuclei). However, depletion of CD8-positive (CD8+) cells resulted in a 40% increase in the number of osteoclasts formed. Addition of CD8+ cells to CD8+ cell depleted cocultures via Transwells abolished the stimulatory effects on osteoclast differentiation resulting from CD8+ cell depletion. Neutralizing antibodies to interleukin-4 and transforming growth factor-beta did not affect osteoclast differentiation in these cultures. These findings suggest that CD8+ cells may be involved in the regulation of osteoclast differentiation and that this effect is not mediated by interleukin-4 or transforming growth factor-beta.

Estrogen and raloxifene stimulate transforming growth factor-beta 3 gene expression in rat bone: a potential mechanism for estrogen- or raloxifene-mediated bone maintenance.

Estrogen or raloxifene (LY156758) prevent estrogen deficiency-induced bone loss in animals and humans. We demonstrated in the rat that a 22% reduction in bone mineral density generated by ovariectomy was associated with a 2-fold reduction of transforming growth factor-beta 3 (TGF beta 3) messenger RNA expression in the femur. Administration of 17 beta-estradiol or raloxifene to ovariectomized rats restored both bone mineral density and TGF beta 3 messenger RNA expression in the femur to levels measured in intact animals. In transient transfection assays, the promoter sequence from -38 to + 110 of the human TGF beta 3 gene, which contains no palindromic estrogen response element, was sufficient to mediate 17 beta-estradiol or raloxifene induced-reporter gene expression in presence of the estrogen receptor. Raloxifene activated TGF beta 3 promoter as a full agonist at nanomolar concentrations. In the same cellular system, raloxifene inhibited the estrogen response element-containing vitellogenin promoter expression as a pure estrogen antagonist. In two well characterized osteoclast differentiation models, TGF beta 3 significantly inhibited the differentiation and bone-resorptive activities of murine and avian osteoclasts. These findings suggest that regulation of TGF beta 3 gene expression by raloxifene or estrogen in bone may be an important target to mediate bone maintenance.

In vivo modulation of murine serum tumour necrosis factor and interleukin-6 levels during endotoxemia by oestrogen agonists and antagonists.

Oestrogen has been reported to modulate tumour necrosis factor (TNF), interleukin (IL)-1 and IL-6 cytokine levels in human mononuclear cell cultures. In the present study, the effects of exogenous oestrogen administration on the cytokine response to an endotoxin challenge was investigated in a murine model of endotoxemia. Animals pretreated for 4 days with 17 alpha ethinyl oestradiol exhibited divergent regulation of TNF and IL-6 levels in sera from endotoxin-stimulated mice. Oestrogen treatment resulted in a significant increase in serum TNF while serum IL-6 levels, relative to the placebo group, decreased in response to an endotoxin challenge. These oestrogenic effects were dose dependent with maximal elevations observed in TNF at 1 mg/kg and maximal reduction in IL-6 at 0.1 mg/kg of 17 alpha ethinyl oestradiol. The increase in TNF levels by ethinyl oestradiol was blocked by co-administration of the oestrogen receptor antagonist tamoxifen. Oestrogen-mediated modulation of the TNF and IL-6 response to endotoxin was also apparent in animals implanted with 17 beta oestradiol pellets. The oestrogen-mediated effects on serum IL-6 were consistent with a reduction in IL-6 mRNA in peritoneal macrophages from oestrogen-treated mice. The effects of oestrogen on TNF and IL-6 production were also investigated in vitro. Oestradiol-treated macrophage cultures produced three- to fourfold lower amounts of IL-6 without any significant modulatory effects on TNF secretion. The combined in vivo and in vitro results demonstrate the modulation of IL-6 and TNF during endotoxemia by oestrogen analogues through an oestrogen receptor-dependent mechanism.

The effect of treating with anti-interleukin-1 receptor antibody on the course of experimental murine cutaneous leishmaniasis.

To assess the role of interleukin-1 (IL-1) in cutaneous leishmaniasis, Leishmania major-infected mice were treated with an anti-IL-1 receptor monoclonal antibody, LA-15.6. MoAb LA-15.6 prevents binding of IL-1 to both the T cell and B cell/macrophage forms of the IL-1 receptor. We found that treating with LA 15.6 inhibited the development of cutaneous lesions of L. major in both genetically-susceptible and resistant mice. Interestingly, this treatment had little or no effect on parasite numbers in the lesions or on the cytokines (interferon-gamma, interleukin-4) that the animals produced in response to infection with the parasite. These results suggest that although IL-1 plays a detrimental role in cutaneous leishmaniasis, it does not mediate this effect by altering the parasite-specific T cell response.

Interleukin-6 secretion from human astrocytoma cells induced by substance P.

Functional NK-1 (substance P) receptors have been demonstrated previously on astrocytes from primary newborn rat brain cultures and human astrocytoma cells lines by specific [125I]-Bolton Hunter substance P (SP) binding and by SP-induced phosphoinositol turnover. In addition, these cells have been shown to release cytokines upon stimulation with interleukin-1 (IL-1) and lipopolysaccharide (LPS). Since SP has also been shown to induce cytokine release from rat glial cells, this neuropeptide may contribute to the pathophysiology of neuronal inflammation in humans by stimulating cytokine production in the brain. We, therefore, explored whether SP could induce U-373 MG human astrocytoma cells, via specific NK-1 receptor activation, to secrete interleukin-6 (IL-6), a cytokine implicated as a key mediator of immune and inflammatory responses. SP stimulated IL-6 production in a concentration-dependent manner with an MC50 (concentration inducing 50% of the maximum response) of 45 nM. IL-6 was detected in the cell culture supernatant fluids 2 h post stimulation and secretion peaked at 12 h. SP induced IL-6 secretion was not mediated by IL-1 since neutralizing anti-IL-1 (alpha and beta) antibody treatment had no effect on the SP response. The selective NK-1 receptor agonist, [Sar9, Met(O2)11]-SP, was comparably effective to SP in stimulating IL-6 secretion; however, selective NK-2 and NK-3 receptor agonists were 250-500-fold less effective. In addition, the non-peptide NK-1 receptor antagonist, (+/-)CP-96,345, inhibited SP (Ki = 4 nM), but not IL-1-induced IL-6 release. These selectivity and specificity studies confirmed the presence of functional NK-1 type receptors linked to IL-6 release. The results of this study support a role for SP as a modulator of immune and/or inflammatory processes in the human CNS.

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Cell proliferation and cytokine production by CD4+ cells from old mice.

Splenocytes from young adult or old C57BL/6NNia mice were stimulated in vitro with the anti-CD3 epsilon mAb, 145-2C11, in either soluble (2C11s) or plate-bound (2C11i) form. In the young group, each mode of cell activation resulted in peak DNA synthesis at approximately 48 h of culture; at this time point, the old group exhibited response levels to 2C11s or 2C11i that were approximately 40% of those in the young group. However, in the presence of 2C11i, splenocytes from old donors showed a delayed peak response which approached the peak levels attained in the young group. To analyze the responsiveness of the CD4+ T cell subpopulation, this cell type was isolated from spleens of young or old mice and was stimulated in vitro with 2C11s or 2C11i, in the presence or absence of added accessory cells (T cell-depleted, irradiated splenocytes). The induction of DNA synthesis by 2C11s was accessory cell dependent, and the response in the old group were markedly reduced in comparison to those in the young group. In contrast, stimulation of DNA synthesis with 2C11i was relatively accessory cell independent, resulted in higher response levels in both age groups, and lessened the disparity between age groups. The analysis of IL-2 and IL-4 secretion by stimulated CD4+ cells revealed that, in response to 2C11s and accessory cells, only IL-2 accumulation was detectable and the levels in the young group were approximately 10-fold higher than the IL-2 levels in the old group. However, stimulation of CD4+ cells with 2C11i and accessory cells yielded improved IL-2 production and a detectable IL-4 response in the old group, whereas the young group exhibited a response profile similar to that induced by 2C11s. Further analysis of the IL-2, IL-4, and IFN gamma mRNA levels in 2C11i-stimulated CD4+ cells revealed that old donor cells accumulated similar levels of IL-2 transcripts, but higher levels of IL-4 and IFN gamma transcripts, than young donor CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)