Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.

Human interleukin-3 mRNA accumulation is controlled at both the transcriptional and posttranscriptional level.

Interleukin-3 (IL-3) is a hematopoietic growth factor that regulates the differentiation of multilineage and committed progenitor cells and the functions of some mature blood cells. The expression of human IL-3 appears to be restricted to stimulated T lymphocytes. We have investigated the kinetics and mechanisms involved in the induction of IL-3 expression in the human T lymphocytic tumor cell line Jurkat. We show that accumulation of IL-3 mRNA is controlled at both the transcriptional and posttranscriptional level. Transcription of the IL-3 gene in these cells appears to be constitutive but no IL-3 mRNA was detected in unstimulated cells, indicating that in resting cells IL-3 mRNA is highly unstable. Treatment with phytohemagglutinin (PHA) induced a small and transient increase in the IL-3 gene transcription rate and led to the production of detectable levels of IL-3 mRNA and protein. Optimal induction of IL-3 expression required a second stimulus. Costimulation of Jurkat cells with both phorbol myristate acetate and PHA caused both a transient increase in IL-3 gene transcription, which is dependent on new protein synthesis, and also a transient increase in mRNA stability.

Synthesis and expression of the gene encoding human interleukin-3.

To perform structure-function studies of human interleukin-3 (hIL-3) we have synthesized a cDNA encompassing the complete coding region of 484 bp. The strategy we employed involved construction of the cDNA in four sections. Each fragment contained six to ten oligodeoxyribonucleotides. Unique restriction sites were engineered to flank the natural sequence for cloning. Naturally occurring restriction sites were placed internally to these, to allow ligation of the four fragments. The gene was cloned into a modified pJL4 vector and expressed in COS cells. Biological assays of supernatants collected from these cells, for both mature cell function and proliferative activity, showed that synthetic hIL-3 had the same activity as that previously determined for recombinant hIL-3.

Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series.

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM-CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.

Effects of quinacrine on vasopressin-induced changes in glycogen phosphorylase activity, Ca2+ transport and phosphoinositide metabolism in isolated hepatocytes.

In isolated hepatocytes, quinacrine (150-250 microM) inhibited vasopressin-induced increases in glucose release, glycogen phosphorylase a activity and 45Ca2+ efflux; and glucagon-induced increases in glucose release and cyclic AMP formation. These results indicate that a phospholipase A2 enzyme sensitive to quinacrine is unlikely to be involved in the process by which vasopressin stimulates glycogen phosphorylase activity in the liver cell. In cells labelled with [3H]inositol, much lower concentrations of quinacrine (20-50 microM) inhibited the stimulation by vasopressin of the accumulation of [3H]inositol. The drug had little effect on vasopressin-induced accumulation of [3H]inositol mono-, bis- and tris-phosphates. In the absence of vasopressin, higher concentrations of quinacrine caused a small stimulation of glycogen phosphorylase activity, 45Ca2+ release and the formation of [3H]inositol polyphosphates. Quinacrine did not inhibit the degradation by liver homogenates of inositol 1-phosphate, inositol 4,5-bisphosphate or inositol 1,4,5-trisphosphate. It is concluded that concentrations of quinacrine comparable with those which inhibit phospholipase A2 [G.J. Blackwell, W.G. Duncombe, R.J. Flower, M.F. Parsons and J.R. Vane, Br. J. Pharmac. 59, 353-366 (1977)] inhibit the stimulation by vasopressin of inositol utilization without significantly affecting coupling between hormone receptors and adenyl cyclase or phosphoinositide-specific phosphodiesterase, the action of the phosphodiesterase, and the degradation of inositol triphosphate.

Studies with verapamil and nifedipine provide evidence for the presence in the liver cell plasma membrane of two types of Ca2+ inflow transporter which are dissimilar to potential-operated Ca2+ channels.

The addition of 500 microM verapamil or nifedipine to isolated hepatocytes incubated in the presence of 1.3 mM Ca2+ caused 20% inhibition of Ca2+ inflow as measured by the initial rate of 45Ca2+ exchange. No stimulation of 45Ca2+ exchange was observed in the presence of the Ca2+ agonist CGP 28392. An increase in the concentration of extracellular K+ from 6 to 60 mM (to depolarize the plasma membrane) increased the initial rate of 45Ca2+ exchange by 30%. In the presence of 60 mM K+, 400 microM verapamil inhibited the initiate rate of 45Ca2+ exchange by 50%. Verapamil and nifedipine completely inhibited vasopressin-induced Ca2+ inflow as determined by measurement of the initial rate of 45Ca2+ exchange and of glycogen phosphorylase a activity. This effect of verapamil was completely reversed by increasing the extracellular concentration of Ca2+. The concentrations of Ca2+ antagonist which gave 50% inhibition of vasopressin- or K+-stimulated Ca2+ inflow were in the range 50-100 microM, about 50-fold greater than the concentration which gave 50% inhibition of the beating of electrically-stimulated myocardial muscle cells. In the absence of vasopressin, verapamil caused a transient increase in glycogen phosphorylase a activity by a process which is largely independent of Ca2+. It is concluded that verapamil and nifedipine inhibit the transport of Ca2+ across the hepatocyte plasma membrane through a putative Ca2+ transporter which is activated by vasopressin and which differs in nature from potential-operated Ca2+ channels in excitable cells and from the Ca2+ transporter present in hepatocytes in the absence of hormone.

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.

Nebraska physicians' attitudes and practices in the field of infant feeding and nutrition.

A questionnaire was mailed to all pediatricians, family practitioners, and general practitioners in Nebraska to determine their attitudes and practices concerning infant feeding and nutrition. One hundred seventy responded. Their attitudes and practices regarding infant feeding and nutrition generally seemed to be positive. Areas in which they were undecided were often nutrition concerns which are currently being researched.

PIP: Purpose of this survey was to investigate physicians' current practices and attitudes on nutritional care and feeding of the infant. The survey was conducted by mailing 426 questionnaires; only 170, or 40% were returned completed and were used. 17% of physicians were pediatricians, and 83% were general practitioners; only 10% had taken specific courses in nutrition in medical school, but most had received some nutrition education. Most physicians were in favor of preventive nutrition, such as taking measures against obesity during infancy; most were unsure of the effects of sodium in infant diets as related to adult hypertension, and of the role of breast feeding and of bottle feeding on weight gain. Most doctors were in favor of breast feeding; 90% recommended adjusting the infant diet in relation to weight; 60% did not recommend a structured feeding schedule; only 41% recommended a prepared formula containing iron, 31% recommended it occasionally, and 21% rarely. 81.7% recommended solid food at or before the 2nd month of age (this percentage was 66% in 1954, and 30% in 1968); 90% recommended cereals by 4 months. 13% recommended discontinuing formula by 3 months, and 73% by 6 months (versus 45% and 92%, respectively, in 1968). 39% of physicians stated that they imparted nutrition information to mothers, while 31% gave printed materials on infant feeding. In general, physicians in this study did not support the debated recommendation for the routine use of iron-fortified milk for infant.