Modulation of human granulopoiesis by macrophage-derived growth regulators

The response of granulocyte progenitors (CFU-D) from patients with chronic myeloid leukaemia (CML), neutrophilic reaction (NR) and healthy subjects to macrophage-derived stimulatory and inhibitory factors was investigated in diffusion chamber culture. CFU-D from CML and NR demonstrated a normal reactivity to macrophage stimulation but were hyporesponsive to indomethacin-sensitive inhibition. It is also shown that the spleens of patients with Hodgkin's disease contain locally activated macrophages with higher production of indomethacin-sensitive growth inhibiting factor for autologous CFU-D clonal proliferation.     

Splenic monocyte-macrophage dependent suppression of autologous granulocyte-progenitors growth in Hodgkin's disease

The studies described compare the effect of spleen cell suspensions from 11 patients with Hodgkin's disease (HD) and 5 healthy subjects on the clonal growth of autologous marrow granulopoietic progenitors in diffusion chamber culture (CFU-G/D). Adherent monocyte/macrophage fraction of splenocytes from HD suppresses the proliferation of autologous CFU-G/D. This inhibition was mediated by an indomethacin-sensitive humoral factor(s). Non-adherent lymphoid cells stimulated myeloid colony formation. Dose response curves demonstrated a markedly increased inhibitory-activity production already by low numbers of splenic monocytes/macrophages from HD whereas a comparable counts of monocytes/macrophages from the spleens of healthy subjects stimulated the CFU-G/D growth. These results may suggest a possible activation of splenic monocytes/macrophages with an enhanced prostaglandin-mediated suppressor activity release for local granulocytopoiesis in the spleens of patients with HD.     

Growth of in vitro colony-forming cells from normal human peripheral blood leukocytes cultured in diffusion chambers

The growth of granulopoietic progenitor cells (CFU-C) in diffusion chambers during culture of peripheral blood leukocytes from 10 normal subjects has been studied. At various times after initiation of diffusion chamber culture, cells harvested from the chambers were transferred to agar culture for measurement of CFU-C concentration. Under these conditions colonies could be grown successfully in agar culture provided pronase, necessary for the chamber harvesting procedure, was first removed by careful washing. A marked increase in the number of CFU-C, up to 25-fold the initial value, was observed in 8 out of 10 subjects. Here the growth pattern was similar, independent of the initial CFU-C values, with an immediate rise to a maximum between 6 and 13 days of culture followed by a decrease. In the other two subjects the growth of CFU-C throughout the diffusion chamber culture period was very poor. The growth of CFU-C from a given individual's blood was shown to be reproducible in repeated studies in 2 subjects, one of whom showed a proliferative and the other a non-proliferative pattern. Evidence suggests that the increase in CFU-C in diffusion chambers is the result of both self-renewal of these cells and influx from a more primitive compartment, although the present data do not allow an estimate of the relative magnitude of each.     

Modulation of normal and abnormal myeloid progenitor proliferation by cyclic nucleotides and PGE1

The effects of cyclic nucleotides and PGE1 upon the proliferation of normal granulocyte/macrophage progenitors were examined in in vitro systems and contrasted to the effects of these compounds on (1) granulocyte/macrophage progenitors from the peripheral blood of patients with myeolofibrosis/myeloid metaplasia (MF) and chronic myelogeneous leukemia (CML); and (2) blast progenitors from the peripheral blood of patients with acute myelogenous leukemia (AML) and acute monocytic leukemia (AMoL). Cyclic AMP was found to be a concentration dependent inhibitor of colony proliferation in all systems tested. Cyclic GMP was an inconsistent enhancer of colony proliferation in all systems in a manner which was not clearly concentration dependent. The effect of PGE1 in normal systems was highly variable depending on the culture conditions, but it was generally found to be an inhibitor of colony proliferation. Cyclic AMP, cyclic GMP and PGE1 altered the release of colony stimulating activity from adherent bone marrow cells in a manner opposite to the direct effects of these compounds on progenitor cell proliferation. Abnormalities in response to PGE1 were found in progenitors from patients with CML (deficient inhibition), AMoL (stimulation of proliferation in certain concentration ranges), and MF (enhanced proliferation). Studies on one of the patients with MF indicated that a normally responding population could be defined by density-gradient separation. These data confirm the capacity of these compounds to modulate in vitro proliferation of myeloid progenitors, and suggest that aberrations of response to PGE1 may occur in subpopulations of cells from several myeloproliferative disorders.     

Scientific Foundations of Oncology

The effect of somatostatin on the thyrotropin (TSH), prolactin, growth hormone (GH) and insulin responses to the combined administration of thyrotropin releasing hormone (TRH) and arginine was studied in six healthy subjects, three hypothyroid patients and three acromegalic patients. Similar inhibition by somatostatin of the TSH and insulin responses was observed in the three groups. While the tetradecapeptide had no significant effect on the prolactin response in the healthy and acromegalic subjects, it caused an unexpected inhibition of the prolactin response in two of the hypothyroid subjects. Contrary to the findings in the healthy and hypothyroid subjects, somatostatin did not inhibit the GH response in the acromegalic patients. Normal inhibition by somatostatin of the insulin response, followed by a rebound in insulin secretion, was observed in all subjects. These preliminary data indicate increased sensitivity of the prolactin-secreting cells to somatostatin in hypothyroidism and suggest that decreased responsiveness of the somatotrophs to somatostatin could play a role in the pathogenesis of acromegaly.     

Responsiveness of plasma aldosterone to angiotensin II in patients with diabetes mellitus

Aldosterone responsiveness to angiotensin II (A II) was evaluated in 65 diabetic patients with and without various diabetic complications versus 38 age-matched non-diabetic subjects. Plasma aldosterone (PA), together with plasma renin activity (PRA), was low and responded poorly to furosemide (80 mg, orally) plus upright posture (4 hours) stimulation in diabetic patients. When the PA response to stimulation relative to PRA response was estimated from the ratio of PA increase to PRA increase after stimulation (delta PA/delta PRA), the 38 non-diabetic subjects had ratios more than 3.0. Of the 65 diabetic patients, 48 had normal delta PA/delta PRA ratios (more than 3.0) and 17 had low delta PA/delta PRA ratios (less than 2.9). Graded A II infusions (1, 2, and 4 ng/kg/min each for 30 min) were performed under a low sodium intake (sodium, 120 mEq/day) in 25 of the 65 diabetic patients, whose delta PA/delta PRA ratios were normal in 15 and low in 10, and in 16 non-diabetic subjects. The PA responses to the graded A II infusions in the normal delta PA/delta PRA diabetic patients were similar to those in the non-diabetic subjects. However, the PA responses to the graded A II infusions in the low delta PA/delta PRA diabetic patients were significantly lower. It is concluded that, although the majority of diabetic patients have normal aldosterone responsiveness to A II, some diabetic patients have blunted aldosterone responsiveness to A II probably attributable to the abnormality of the adrenal cortex in addition to the impaired renin secretion.     

Usefulness of the assessment of erythroid progenitors growth for differential diagnosis of primary and secondary polycythemias

Erythropoietic progenitors from bone marrow of patients with polycythemia vera (PV), secondary polycythemia (SP) and healthy subjects (HS) were cultured in plasma clot diffusion chambers in vivo. The chambers were inserted into the peritoneal cavities of rats, which 24 and 2 h before implantation received an injection of phenylohydrazine. Control experiments were done without erythropoietin (Epo) stimulation. Colonies after 2 and 7 days of culture were considered to be formed by mature erythropoietic progenitors (CFU-D-E) and burst forming cells (BFU-D-E), respectively. PV-erythroid progenitors, both BFU-D-E and CFU-D-E produced markedly more colonies than those from SP and HS, especially in experiments without Epo stimulation (p less than 0.01). The plating efficiency in SP was comparable to that noted in HS (p greater than 0.05). These results have led us to postulate that the study of erythroid progenitor clonal proliferation in plasma clot diffusion chamber can be helpful in the differential diagnosis of PV and SP, when other clinical and laboratory findings are not sufficiently convincing.     

Response of Leucocyte Adenyl Cyclase to Isoprenaline and Effect of Alpha-blocking Drugs in Extrinsic Bronchial Asthma

The finding by several workers that biochemical responses to catecholamines are diminished in asthmatic patients during periods of active asthma as compared to normal subjects has led to the recognition of the beta-adrenergic blockade phenomenon, a common accompaniment of extrinsic bronchial asthma. Using an intact cell method to measure leucocyte adenyl cyclase activity, we have been able to show that there is a noticeably reduced responsiveness of this enzyme system (which is now identified with beta-receptor function) to isoprenaline in the leucocytes of patients suffering from acute bronchial asthma, but that asthmatic patients in remission could not be distinguished from normal persons in this respect. Evidently the defective beta-receptor function may be associated with overactivity of the alpha-receptors in acute bronchial asthma, since the responsiveness to isoprenaline stimulation could be restored towards normal by concomitant treatment of the leucocytes of these patients with alpha-receptor blocking drugs such as phentolamine or thymoxamine. Ouabain, though somewhat less potent, also enhanced responsiveness to isoprenaline stimulation. The relation of these results to the clinical observation of adrenaline resistance in active asthma suggests that alpha-receptor blocking drugs may be of value in restoring the sensitivity of beta-receptors to sympathomimetic amines.     

Comparison of immune responsiveness in mice after single or multiple donor-specific transfusions

Immune responsiveness was compared in B6AF1 mice after one, two, three, or four donor-specific DBA/2 blood transfusions (DST). Ten days after the last transfusion, the spleen cells of transfused mice were assayed for direct lymphocyte-mediated cytotoxicity, for the ability to respond in mixed lymphocyte culture (MLC) and cell-mediated lymphocytotoxic (CML) assays to DBA/2 and C3H/He antigens, and for the ability to inhibit the MLC and CML response of normal B6AF1 to DBA/2 and C3H/He antigens. Immune responsiveness was also tested in B6AF1 2 to 80 days after a single DBA/2 DST. The MLC response of transfused mice was specifically suppressed to the blood donor after both single and multiple transfusions. The CML response to DBA/2 was suppressed after a single DST, but returned to normal after multiple transfusions. Spleen cells from transfused mice did not inhibit the MLC response of normal B6AF1 mice to DBA/2 or C3H/He antigens after one or two transfusions regardless of time tested, but were able to inhibit the response to both stimulators after three or more transfusions. The MLC response remained specifically suppressed to the blood donor for as long as 80 days after a single DST, while the CML response was suppressed up to 50 days after transfusion, but had returned to normal by 80 days.     

Inhibition of endotoxin-induced macrophage chemokine production by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in vitro and in vivo.

Inflammatory chemokines recruit various populations of immune cells that initiate and maintain the inflammatory response against foreign Ags. Although such a response is necessary for the elimination of the Ag, the inflammation has to be eventually resolved in a healthy organism. Neuropeptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released after antigenic stimulation, contribute to the termination of an inflammatory response primarily by inhibiting the production of proinflammatory cytokines. Here we investigated the effects of VIP and PACAP on chemokine production. We report that VIP and PACAP inhibit the expression of the macrophage-derived CXC chemokines macrophage inflammatory protein-2 and KC (IL-8), and of the CC chemokines MIP-1alpha, MIP-1beta, monocyte chemoattractant protein 1, and RANTES in vivo and in vitro. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NF-kappaB binding and transactivating activity. The VIP/PACAP inhibition of both chemokine production and of NF-kappaB binding and transactivating activity is mediated through the specific VIP receptor VPAC1, and involves both cAMP-dependent and -independent intracellular pathways. In an in vivo model of acute peritonitis, the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the recruitment of polymorphonuclear cells, macrophages, and lymphocytes into the peritoneal cavity. These findings support the proposed role of VIP and PACAP as key endogenous anti-inflammatory agents and describe a novel mechanism, i.e., the inhibition of the production of macrophage-derived chemokines.     

Natural killer cell immunodeficiency in patients with chronic myelogenous leukemia. IV. Interleukin-1 deficiency, gamma-interferon deficiency and the restorative effects of short-term culture in the presence of interleukin-2 on natural killer cytotoxicity, natural killer-target binding and production of natural killer cytotoxic factor

We report herein that defective natural killer (NK) cell cytotoxicity, NK cytotoxic factor (NKCF) production and NK target binding ability of patients with chronic myelogenous leukemia (CML) are functionally restorable after short-term culture (less than 1 week) with recombinant interleukin-2 (rIL-2). We have previously reported that, despite normal to increased numbers of CD16+ large granular lymphocytes, fluorescence-activated-cell-sorted NK cells from CML patients are profoundly defective in NK cell activity and are unable to lyse the CML blast-crisis-derived, NK-sensitive target K562. Since we and others have also previously shown that the defective NK cytotoxicity from CML patients is restorable after 1-4 weeks of incubation with rIL-2, we therefore deemed it important to study the kinetics of IL-2-mediated NK restoration at earlier time intervals (less than 1 week). In the present report, we have demonstrated a significant restoration of NK cell cytotoxicity in CML patients against K562 after 5 days of short-term culture with rIL-2. In addition, recovery of NKCF production and restoration of target-binding capacity to normal levels by NK cells from CML patients were also observed after short-term (less than 1 week) rIL-2 treatment. Finally, we have demonstrated in the present report that adherent cells and peripheral-blood lymphoid cells from CML patients, as compared to normal controls, are unable to produce IL-1 beta and interferon-gamma, respectively, after stimulation with phorbol myristate acetate (IL-1 beta) and phytohemagglutinin-M (interferon-gamma).     

The mechanism of aldosterone response to furosemide test in patients with Shy-Drager syndrome

We examined the renin-angiotensin-aldosterone system in seven patients with Shy-Drager syndrome by studying their response to the stimulation of 1 mg/kg furosemide injection followed by sitting for 1 hour. Six of the seven patients showed a low response of plasma renin activity to the stimulation. However, in five of the low responders, the plasma aldosterone levels after stimulation were observed to be similar to those of the control subjects; in addition, an increment in the plasma cortisol level appeared although no such increment was observed in normal subjects. Next, we studied the aldosterone response to angiotensin II. The five patients who showed a low plasma renin activity response and a normal aldosterone response to furosemide administration also showed low plasma aldosterone response to angiotensin II. Furthermore, in the patients who demonstrated a low plasma renin activity response and a normal aldosterone response to furosemide administration, the pretreatment with 2 mg dexamethasone for 2 days caused a marked inhibition of aldosterone response to the stimulation. These findings suggested that in most patients with Shy-Drager syndrome, the plasma aldosterone response to the stimulation of furosemide injection followed by sitting for 1 hour might be controlled by ACTH but not by plasma renin activity.     

Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGFβ-release in response to cyclic strain

Mechanical stimulation of bone tissue by physical activity stimulates bone formation in normal bone and may attenuate bone loss of osteoporotic patients. However, altered responsiveness of osteoblasts in osteoporotic bone to mechanical stimuli may contribute to osteoporotic bone involution. The purpose of the present study was to investigate whether osteoblasts from osteoporotic patients and normal donors show differences in proliferation and TGFβ production in responses to cyclic strain. Human osteoblasts isolated from collagenase-treated bone explants of 10 osteoporotic patients (average age 70 ± 6 yr) and 8 normal donors (average age 54 ± 10 yr) were plated into elastic rectangular silicone dishes. Subconfluent cultures were stimulated by cyclic strain (1%, 1 Hz) in an electromechanical cell stretching apparatus at three consecutive days for each 30 min. The cultures were assayed for proliferation, alkaline phosphatase activity and TGFβ release in each three parallel cultures. In all experiments, osteoblasts grown in the same elastic dishes but without mechanical stimulation served as controls. Significant differences between stimulated cultures and unstimulated controls were determined by a paired two-tailed Wilcoxon test. In comparison to the unstimulated controls, osteoblasts from normal donors significantly increased proliferation (p = 0.025) and TGFβ secretion (p = 0.009) into the conditioned culture medium. In contrast, osteoblasts from osteoporotic donors failed to increase both proliferation (p > 0.05) and TGFβ release (p > 0.05) in response to cyclic strain. Alkaline phosphatase activity was not significantly affected (p > 0.05) in normal as well as osteoporotic bone derived osteoblasts.

These findings suggest a different responsiveness to 1% cyclic strain of osteoblasts isolated from normal and osteoporotic bone that could be influenced by both the disease of osteoporosis and the higher average age of the osteoporotic patient group. While osteoblasts from osteoporotic donors failed to increase proliferation and TGFβ release under the chosen mechanical strain regimen that stimulated both parameters in normal osteoblasts, it is possible that some other strain regimen would provide more effective stimulation of osteoporotic cells.     

Influence of central dopaminergic activity on thyrotropin responsiveness to thyrotropin-releasing hormone in normal and thyrotoxic subjects

To study whether central dopaminergic activity influences TSH responsiveness to TRH in normal individuals and in patients with hyperthyroidism, three experiments (A, B and C) were carried out in 8 normal subjects, and two experiments (A and B) in 8 patients with untreated thyrotoxicosis. In experiment A oral placebo (PBO) preceded iv administration of 200 micrograms TRH by 90 min. In experiment B dopamine receptor blockade with 15 mg oral metoclopramide (MET) was given 90 min before iv administration of 200 micrograms TRH. In experiment C two oral doses (each dose 2.5 mg) of bromocriptine (BCT), known for dopamine agonistic properties, were given 9 and 1 hour before ingestion of 15 mg MET which, in turn, preceded iv injection of 200 micrograms TRH by 90 min. In the healthy subjects experiment A revealed a TSH responsiveness, as reflected by the TSH incremental area, which was 430 +/- 74. The corresponding TSH responsiveness was significantly larger in experiment B (661 +/- 138; P less than 0.02). In experiment C the TSH incremental area (332 +/- 102) did not differ significantly from the one obtained in experiment A. The thyrotrophs responded quite different to TRH in the group of thyrotoxic patients, where the TSH incremental area was zero regardless of whether PBO or MET were given as oral pretreatments. These results imply that central dopaminergic activity inhibits the pituitary thyrotrophs and modulates the TSH response to TRH in healthy subjects, but does not contribute significantly to the blocked TSH responsiveness in patients with untreated hyperthyroidism.     

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

Actin polymerization in response to different chemoattractants is reduced in granulocytes from chronic myeloid leukemia patients

Chronic myeloid leukemia (CML), a hematopoietic stem cell disorder, is characterized by the presence of Philadelphia chromosome (Ph1). Earlier studies have shown that various functions, such as chemotaxis, fluid phase pinocytosis, phagocytosis, and degranulation in response to chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), were defective in polymorphonuclear leukocytes (PMNL) from CML patients. These functions depend on actin microfilaments (MF). Further studies showed that fMLP-induced actin polymerization was lower in CML PMNL. To see if this defect is specific to stimulation by fMLP alone or is a global phenomenon involving other chemoattractant receptors, chemotaxis and actin polymerization were studied in response to fMLP, an analog of fMLP, formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenyalanine-O-methionine (FACC8), platelet-activating factor (PAF), and leukotriene B4 (LTB4). These compounds bind to different chemoattractant receptors. Chemotaxis and actin polymerization in response to all four chemoattractants were significantly lower in CML PMNL compared with PMNL from normal subjects and were differentially affected for the different chemoattractants. These results suggest a global phenomenon involving all four chemoattractant-stimulated pathways. This lower amount of F-actin may be responsible for the defective chemotaxis seen in these cells.     

TCR-Independent Induction of Low Responsiveness by Chemically Fixed Cells in Alloreactive CTL Clones and Its Prevention through Cognate Cell-Cell Interaction

We established BALB/c-derived CD8⁺ CTL clones D2-22 (V_β 6⁺), D2-23 (V_β 8⁺) and D2-24 (V_β 8⁺) specific for B10.D2 minor H antigen. D2-22 and D2-23 proliferated without producing IL-2 in response to X-ray-irradiated antigenic cells, Con A, aCD3, PMA and IL-2. Paraformaldehyde-fixed antigenic spleen cells neither induced proliferation in the presence of costimulatory cells nor inhibited responses to irradiated antigenic cells added simultaneously. Unlike the previously reported results with IL-2-producing CTL clones and Th1 clones, the fixed antigenic cells failed to induce antigen-specific unresponsiveness in these IL-2-nonproducing CTL clones. Instead, the responsiveness of these clones to fresh stimulation was found to be reduced severely after 2 days in the culture added with either antigenic or syngeneic fixed cells. Induction of their antigen-nonspecific low responsiveness by the fixed cells was prevented by adding irradiated syngeneic cells into the culture or even by increasing the concentration of responder D2-23 cells. Close contact of D2-23 and irradiated syngeneic cells was required to prevent the reduction of the responsiveness, although this cognate cell-cell interaction could be replaced by exogenously added IL-2 or PMA. Cytolytic and tumor cell growth inhibitory activities of D2-23 were also reduced by incubation with the fixed cells, which was prevented by the addition of irradiated syngeneic cells. These findings showed the unique properties of IL-2-nonproducing CTL clones in signal requirements for maintaining normal responsiveness for proliferation and cytolytic activity.     

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.     

Effect of insulin receptor down regulation on insulin-stimulated thymidine incorporation in cultured human fibroblasts and tumor cell lines.

Insulin receptors in transformed tissue are relatively resistant to down regulation by insulin, and although receptor downregulation reduces rapid onset biologic responses to insulin in normal tissue, this is not observed in tumor cells. The present study compares longterm insulin responses (thymidine incorporation and cell growth) in normal human fibroblasts with responses in human tumor cell lines (MCF-7, T-47D and HCT-8) to determine whether these responses are also resistant to the effects of receptor down regulation. Thymidine incorporation into fibroblasts was more responsive to insulin than was incorporation into tumor cells, although stimulation of uptake into fibroblasts was not paralleled by changes in cell replication. In contrast, physiological insulin concentrations inhibited, and high concentrations of insulin stimulated, thymidine incorporation and cell replication in MCF-7 and T-47D cells. All insulin concentrations inhibited thymidine incorporation in HCT-8 cells without affecting cell replication. The responsiveness of fibroblasts, MCF-7 and HCT-8 cells to insulin was unaltered by down regulation of insulin receptors prior to measuring thymidine incorporation, whereas receptor down regulation paradoxically increased the responsiveness of T-47D cells to insulin. Exposure of fibroblasts to 5 x 10(-8) M dexamethasone for 24h increased their responsiveness to insulin but did not influence the response of MCF-7 or HCT-8 cells, whereas insulin-stimulated incorporation of thymidine in T-47D cells was inhibited. Thus, receptor down regulation does not influence the longterm biologic response to insulin in normal cells, and paradoxically increases responsiveness in one of three tumor cell lines. These changes may contribute to the well-described stimulatory effects of insulin on tumor cell growth and inhibition of this response with dexamethasone may be relevant to cancer treatment programs.