Number Of High-affinity Receptors Research Articles

The Effector Function of Allergens

Allergens are antigens that generate an IgE response (sensitization) in susceptible individuals. The allergenicity of an allergen can be thought of in terms of its ability to sensitize as well as its ability to cross-link IgE/IgE receptor complexes on mast cells and basophils leading to release of preformed and newly formed mediators (effector activity). The identity of the allergens responsible for sensitization may be different from those that elicit an allergic response. Effector activity is determined by (1) the amount of specific IgE (sIgE) and in some circumstances the ratio of sIgE to total IgE, (2) the number of high affinity receptors for IgE (FcεR1) on the cell surface, (3) the affinity of binding of sIgE for its epitope and, in a polyclonal response, the collective avidity, (4) the number and spatial relationships of IgE binding epitopes on the allergen and (5) the presence of IgG that can bind to allergen and either block binding of sIgE and/or activate low affinity IgG receptors that activate intracellular inhibitory pathways. This review will discuss these important immunologic and physical properties that contribute to the effector activity of allergens.

Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2.

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.

Gender-dependent effect of ageing on peripheral insulin action

The aim of the present study was to investigate the effect of both gender and age on insulin secretion, peripheral insulin effectiveness and insulin-receptor binding. Eighty healthy volunteers, 40 females of mean age 38.47 +/- 11.37 years and mean BMI 21.99 +/- 2.06 kg/m(2) and 40 males of mean age 34.87 +/- 11.22 years and mean BMI 22.65 +/- 2.31 kg/m(2), with normal glucose tolerance participated in the study. Peripheral insulin effectiveness was measured by the artificial endocrine pancreas, using the euglycaemic hyperinsulinaemic clamp technique and insulin-receptor binding on circulating mononuclear blood cells. Peripheral insulin sensitivity was significantly higher in females as compared to males (p < 0.001), while males demonstrated higher total number of insulin receptors (p < 0.0001) and number of high-affinity receptors (p < 0.01). Peripheral insulin sensitivity decreased with ageing in both males and females, the reduction in females being more pronounced (p < 0.05). In the group under 40 years, the females demonstrated significantly higher insulin sensitivity as compared to males (p < 0.001) and lower insulin-receptor binding. Over 40 years, females presented higher peripheral insulin sensitivity and higher insulin-receptor binding. The percentage of specifically bound insulin increased significantly with ageing in females and decreased in males. We consider that probably the higher androgen level in males affects the post-receptor processes in insulin action and despite the higher insulin-receptor binding, males have lower insulin sensitivity. The androgen levels in females increase with ageing, which could probably affect peripheral insulin sensitivity at the post-receptor level. In conclusion, our results demonstrate that when analysing peripheral insulin effectiveness and insulin-receptor binding, one should always consider both gender and age.

Appearance Of A Novel Prostacyclin Receptor Antibody and Duration Of Spinal Cord Injury

Background: Cardiovascular disease (CVD) appears to be accelerated in individuals with chronic spinal cord injury (SCI). Previously, we have identified a novel circulating antibody (lgG) in persons with SCI that specifically blocks the high-affinity prostacyclin (PGI) receptors on the platelet surface without affecting the low-affinity PGI 2 receptors.Objective: In this study, the relationship between the time course after SCI to the development of lgG to the high-affinity PGI 2 receptor was determined.Methods: Blood samples were collected 1, 3, 5, 10, and > 1 0 (15 ±4) years after SCI (n = 36). Plasma samples (50 11g) were analyzed by polyacrylamide gel electrophoresis (PAGE)followed by ensitometry.Results: The optical density (OD) of the lgG (molecular weight 47,000) at 1 year afterSCI was significantly higher than control (1 .65 ±0.08 vs 1.33 ±0.04; P < 0.01 ). Thisanti-receptor lgG appears to increase for 5 years and then plateau. At 5 years, 6-10 years,and > 10 years of injury, the OD was 1.83± 0.09, 1.83 ±0.1 0, and 1.87 ±0.08, respectively. With an increase in this specific lgG, there was a concomitant decrease in the binding of prostacyclin to its high-affinity receptors on SCIplatelets, (non-SCI vs 1, 3, and5years after injury; n1 = 1 72 ±25 vs 153 ±15, 1 07 ±25, and 40 ±4 sites/platelet, respectively; P < 0.001 ), with no significant change inreceptor affinity.Conclusions: The level of the high-affinity PGI2-receptor antibody determined in individuals with SCI was associated with the duration and not with the level of injury. Plateletsfrom subjects with SCI had a reduction in numbers of high-affinity receptors.

An Extracellular Protein Microdomain Controls Up-regulation of Neuronal Nicotinic Acetylcholine Receptors by Nicotine

In smoker's brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.

Biochemical and molecular biological aspects of glucose intolerance in elderly persons

Changes in carbohydrate metabolism in elderly persons have drawn considerable attention but the findings from different studies are in contrast and are even controversial. The insulin receptors in erythrocytes were studied in elderly euglycaemic patients and in a middle-aged control group. The examined persons were also subjected to measurements of blood glucose, insulin and C-peptide concentrations, before and 3 hours after a dietetic meal. In the present study it was found that in spite of the maintained insulin level and C-peptide secretion, some structural and corresponding changes in membrane insulin receptors and the binding site caused differences in postreceptor responses in elderly persons. The examined groups, consisted of 29 males, average age of 66 years (65-70), with normal serum glucose level and 19 middle-aged males, average age of 42 years (32-48), with normal glycoregulation. In basal condition, elderly persons have both normal morning serum insulin (19.68 ? 4.00 mU/L) and C-peptide (2.04 ? 0.78 nmol/L) level. In elderly persons, the number of high affinity insulin receptors in erythrocytes membrane is 22.80 ? 6.18 but the formed insulin-high affinity receptors were not stable. Dissociation constant (Kd1) indicates its elevated dissociation (0.11 ? 0.04). At the same time the number of insulin low affinity binding sites is increased (13 273 ? 5 572) with a fast dissociation of the hormone (13.99 ? 3.37). Food intake raised the number of high affinity receptors compared to the basal value. Alteration in insulin binding affinity suggests the structural and corresponding changes in membrane receptors that may cause differences in postreceptors responses in elderly persons.

Modulation of androgen receptor protein by culture conditions of human skin fibroblasts

Cultures of skin fibroblasts show variation of androgen binding with culture conditions; binding variations are usually avoided by using confluent cultures. In this work, we analysed the effect of cell density and mitogenic agents on the level of androgen receptor (AR) of cultured human skin fibroblasts. Results demonstrated that in cultures of human skin fibroblasts, cellular binding of dihydrotestosterone was higher in cells grown at low than at high cell density. The reduction in binding resulted from a decrease in the number of high affinity receptors and not from a change in receptor affinity. Immunocytochemistry for AR showed greater staining intensity in cells grown at low than at high cell density. Additionally, immunoblot analysis demonstrated more AR protein in low cell density cultures. On the other hand, it was observed that cells grown at low cell density showed diminished androgen binding capacity after 24 h of treatment with insulin-like growth factor (IGF-l), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or granulocyte-colony stimulating factor (G-CSF); this effect of growth factors was not observed in cells grown at high cell density. In conclusion, we found that cell density of cultures and mitogenic agents can regulate AR binding activity in human fibroblasts. While we do not yet know how changes in cell density affect the amount of AR, we conclude that the mechanism could be mediated by activation of the tyrosine kinase pathway, as the effect was reproduced by mitogens.

Evidence for Multiple Rat VPAC 1 Receptor States with Different Affinities for Agonists

We compare the binding properties of [ 125I-VIP] and [ 125I]-Ro 25 1553 to VPAC 1 receptors, expressed in stably transfected CHO cells. [ 125I]-VIP labelled two VPAC 1 receptor states, while [ 125I]-Ro 25 1553 labelled selectively a limited number of high-affinity receptors. This high-affinity state probably corresponds to an agonist-receptor-G s ternary complex as its properties (guanyl nucleotides, EC 50 values and maximal effect) were affected by cholera toxin pre-treatment. Both high- and low-affinity receptors participated in the adenylate cyclase activation. This suggested that agonists activate not only low-affinity uncoupled receptors by facilitating the ternary complex formation, but also activated the high-affinity ternary complex by accelerating the GTP binding to emptied, receptor-bound G proteins.

Homologous Up-regulation of KDR/Flk-1 Receptor Expression by Vascular Endothelial Growth Factor in Vitro

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

Inhibition of the growth of Caki-I human renal adenocarcinoma in vivo by luteinizing hormone-releasing hormone antagonist Cetrorelix, somatostatin analog RC-160, and bombesin antagonist RC-3940-II.

Metastatic or recurrent renal cell carcinoma (RCC) is a therapeutic challenge because it is resistant to chemotherapy and external radiotherapy. No uniformly effective therapeutic agents are available for the management of patients with RCC. Hormones and growth factors may play a role in promoting the transformation and/or proliferation of kidney neoplasms. Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix (SB-75), somatostatin analog RC-160, and bombesin antagonist RC-3940-II were tested for their effects on the growth of the Caki-I renal adenocarcinoma cell line xenografted into nude mice. After 4 weeks of treatment, tumor volume was significantly (P < 0.01) decreased in animals receiving RC-160, to 167.5 +/- 34.2 mm3, compared with the control group (485.7 +/- 77.2 mm3). LH-RH antagonist SB-75 and bombesin antagonist RC-3940-II also significantly reduced the volume of Caki-I tumors, to 159.9 +/- 18.1 and 234.7 +/- 81.8 mm3, respectively. Somatostatin analog RC-160 decreased serum levels for growth hormone (GH) and insulin-like growth factor-I compared with controls. Treatment with RC-160, Cetrorelix, and RC-3940-II significantly reduced the number of high-affinity receptors for epidermal growth factor on Caki-I tumors. LH-RH antagonist Cetrorelix, somatostatin analog RC-160, and bombesin antagonist RC-3940-II effectively inhibit the growth of human Caki-I renal adenocarcinomas in nude mice. These peptide analogs should be considered for the therapy of patients with metastatic or recurrent RCC.

Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy.

We have detected the binding of the fluorescent agonist Dns-C6-Cho to both low- and high-affinity states of the nicotinic acetylcholine receptor (nAcChoR) using sequential mixing stopped-flow fluorescence spectroscopy. Our approach to resolving low- and high-affinity binding was to first preincubate receptor membranes with the fluorescent partial agonist Dns-C6-Cho for 15 ms to 1000 s and then to follow the fluorescence decay upon chemical dilution into excess acetylcholine. The fast and slow decays, reflecting Dns-C6-Cho dissociation from low- and high-affinity receptors, had rates of 140 +/- 27 s-1 and 0.1 +/- 0.02 s-1, respectively. With increasing preincubation times, the number of low-affinity receptors decreased while the number of high-affinity receptors increased in a Dns-C6-Cho concentration-dependent manner consistent with current models for agonist-induced affinity state conversion. At receptor-activating concentrations of Dns-C6-Cho, the apparent rates with which high-affinity receptors formed approximated those of ion flux desensitization, implying that the fast desensitized state has an agonist dissociation rate that is indistinguishable from the equilibrium slow desensitized state. The KD for the low-affinity binding site was determined to be 1.1 microM from the increase in the amplitude of the fast decay with Dns-C6-Cho concentration with preincubation times that were sufficiently brief to minimize affinity state conversion. Assuming a bimolecular association rate of 10(8) M-1 s-1, a second estimate of 1.4 microM was made for low-affinity binding. We also detected a fluorescence enhancement consistent with a conformational isomerization of Dns-C6-Cho-inhibited nAcChoRs.

Modulation of the Apoptotic Response of Human Myeloid Leukemia Cells to a Diphtheria Toxin Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractIt has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor–bearing cells. We now report that DT388–GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF–receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL ). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I–GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388–GM-CSF at 4 × 10−9 mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 × 10−9 mol/L to 3 × 10−13 mol/L. GM-CSF–receptor occupancy at 0.7 nmol/L GM-CSF–ligand concentration correlated with the protein synthesis IC50 . Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL , in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388–GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.

In vitro and in vivo vascular actions of basic fibroblast growth factor (bFGF) in normotensive and spontaneously hypertensive rats.

In the cardiovascular system, basic fibroblast growth factor (bFGF) is an important modulator of blood vessel growth and blood pressure and, as such, could contribute to the structural and functional changes that contribute to hypertension. This study evaluated in vitro and in vivo vascular actions of bFGF in normotensive and hypertensive rats. Basic FGF increased the expression of the messenger RNA (mRNA) encoding for the immediate early gene, egr-1, in cultured vascular smooth muscle cells (VSM) isolated from normotensive Wistar-Kyoto (WKY). Sprague-Dawley (SD), and spontaneously hypertensive rats (SHRs). Maximal increases (stimulated/control) in egr-1 mRNA were greater in SHR than in WKY and SD rat VSM (14.57 +/- 1.94 vs. 5.75 +/- 0.35 and 3.84 +/- 0.70). Basic FGF (30 ng/ml) stimulated the growth of WKY (43 +/- 8.4% of growth in 10% serum) and SD (34.6 +/- 6.5%) rat and SHR (75.8 +/- 8.8%) VSM but was most efficacious at stimulating SHR VSM. Radioligand-binding assays indicated no differences in the affinity or number of high-affinity receptors but that the binding of bFGF to low-affinity receptors was slightly but significantly greater in SHR VSM. In vivo, bFGF vasodilated cremaster arterioles in normotensive but not in hypertensive rats. These data suggest that hypertensive animals are more responsive to the growth-stimulatory actions but are less responsive to the vasodilatory actions of bFGF. This altered bFGF function could contribute to the development or maintenance or both of hypertension.

Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury.

Coronary artery disease is a leading cause of death in individuals with chronic spinal cord injury (SCI). However, platelets of those with SCI (n = 30) showed neither increased aggregation nor resistance to the antiaggregatory effects of prostacyclin when compared with normal controls (n = 30). Prostanoid-induced cAMP synthesis was similar in both groups. In contrast, prostacyclin, which completely inhibited the platelet-stimulated thrombin generation in normal controls, failed to do so in those with SCI. Scatchard analysis of the binding of [3H]prostaglandin E1, used as a prostacyclin receptor probe, showed the presence of one high-affinity (Kd1 = 8.11 +/- 2.80 nM; n1 = 172 +/- 32 sites per cell) and one low-affinity (Kd2 = 1.01 +/- 0.3 microM; n2 = 1772 +/- 226 sites per cell) prostacyclin receptor in normal platelets. In contrast, the same analysis in subjects with SCI showed significant loss (P < 0.001) of high-affinity receptor sites (Kd1 = 6.34 +/- 1.91 nM; n1 = 43 +/- 10 sites per cell) with no significant change in the low affinity-receptors (Kd2 = 1.22 +/- 0.23; n2 = 1820 +/- 421). Treatment of these platelets with insulin, which has been demonstrated to restore both of the high- and low-affinity prostaglandin receptor numbers to within normal ranges in coronary artery disease, increased high-affinity receptor numbers and restored the prostacyclin effect on thrombin generation. These results demonstrate that the loss of the inhibitory effect of prostacyclin on the stimulation of thrombin generation was due to the loss of platelet high-affinity prostanoid receptors, which may contribute to atherogenesis in individuals with chronic SCI.

Effect on tumorigenicity and metastasis of transfection of a diploid benign rat mammary epithelial cell line with DNA corresponding to the mRNA for basic fibroblast growth factor

To examine the potential role of fibroblast growth factors (FGF) in tumorigenesis and metastasis, plasmid constructs containing the human basic FGF (bFGF) gene, with or without fusion to a secretory signal peptide (IgbFGF), were transfected into the diploid rat mammary epithelial cell line Rama 37. All transfectants possessed multiple copies of the transfected cDNA, which was expressed as the corresponding mRNA and the protein. The amount of bFGF protein was usually greater than the bFGF growth-stimulatory activity that could be recovered from the transfected cells. Nevertheless, the amount of bFGF growth-stimulatory activity secreted by the IgbFGF transfectants (0.08-0.8 ng/ml/24 hr) was sufficient to induce growth in responsive cells. However, the transfectants themselves were refractory to stimulation by exogenously added bFGF, despite possessing a small number of high-affinity receptors for bFGF. When the bFGF or the IgbFGF transfectants were inoculated into the mammary fat pads of syngeneic rats, the tumour incidence was low (0-50%). However, when cells cultured from these tumours were inoculated into the fat pad of syngeneic rats, the tumour incidence was 100%. Tumours were in all cases benign and no metastases were observed. Our results suggest that the role of bFGF in metastasis is not simply one of autocrine/paracrine stimulation of cell growth and that other events may also be required.

Effect on tumorigenicity and metastasis of transfection of a diploid benign rat mammary epithelial cell line with DNA corresponding to the mRNA for basic fibroblast growth factor

To examine the potential role of fibroblast growth factors (FGF) in tumorigenesis and metastasis, plasmid constructs containing the human basic FGF (bFGF) gene, with or without fusion to a secretory signal peptide (IgbFGF), were transfected into the diploid rat mammary epithelial cell line Rama 37. All transfectants possessed multiple copies of the transfected cDNA, which was expressed as the corresponding mRNA and the protein. The amount of bFGF protein was usually greater than the bFGF growth-stimulatory activity that could be recovered from the transfected cells. Nevertheless, the amount of bFGF growth-stimulatory activity secreted by the IgbFGF transfectants (0.08–0.8 ng/ml/24 hr) was sufficient to induce growth in responsive cells. However, the transfectants themselves were refractory to stimulation by exogenously added bFGF, despite possessing a small number of high-affinity receptors for bFGF. When the bFGF or the IgbFGF transfectants were inoculated into the mammary fat pads of syngeneic rats, the tumour incidence was low (0–50%). However, when cells cultured from these tumours were inoculated into the fat pad of syngeneic rats, the tumour incidence was 100%. Tumours were in all cases benign and no metastases were observed. Our results suggest that the role of bFGF in metastasis is not simply one of autocrine/paracrine stimulation of cell growth and that other events may also be required. © 1996 Wiley-Liss, Inc.

Fast and high-affinity binding of B-lymphotropic papovavirus to human B-lymphoma cell lines.

Binding of B-lymphotropic papovavirus (LPV) to host cells differing in susceptibility to viral infection was determined by a newly established, direct, nonradioactive virus binding assay, which allows quantitative description of the binding characteristics by receptor saturation and Scatchard analysis. LPV binding to the highly susceptible human B-lymphoma cell line BJA-B K88 is specific, saturable, and noncooperative. Binding occurs very fast, with an association rate constant (k1) of 6.7 x 10(7) M-1s-1, and is of high affinity, with a dissociation constant (Kd) of 2.9 x 10(-12) M; and the virus-receptor complex is stable, with a half life of 70 min. The binding affinities of receptors on four other highly, moderately, or weakly susceptible human B-lymphoma cell lines were similar, with up to twofold variation around a mean Kd value of 3 x 10(-12) M, suggesting the presence of the same LPV receptor on all of these cell lines. This view is further supported by the finding that in all cases a terminal sialic acid is necessary for LPV binding. Tunicamycin has been shown to drastically induce LPV susceptibility and LPV binding in weakly and moderately susceptible B-lymphoma cell lines (O.T. Keppler, M. Herrmann, M. Oppenländer, W. Meschede, and M. Pawlita, J. Virol. 68:6933-6939, 1994). The hypothesis that the constitutively expressed and tunicamycin-induced LPV receptors are identical is strengthened by our finding that both receptor types displayed the same high affinity. LPV susceptibility of different B-lymphoma cell lines was correlated with receptor number but not with receptor affinity. The numbers of receptors per cell on highly and moderately susceptible cell lines ranged from 2,000 to 400 and were directly proportional to LPV susceptibility. This indicates that the number of high-affinity receptors per cell is a key regulating factor for the LPV host range.

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Substance P (SP) Mediates Production of Stem Cell Factor and Interleukin-1 in Bone Marrow Stroma: Potential Autoregulatory Role for These Cytokines in SP Receptor Expression and Induction

Substance P (SP) is a neuropeptide widely distributed in the nervous system. Its release within the bone marrow (BM) can mediate bidirectional neurohematopoietic communication via specific receptors: neurokinin-1R (NK-1R), NK-2R, or NK-3R. We have previously reported that SP effects on hematopoiesis are mediated by an NK-1-type receptor, the BM stroma, and growth factors. Here, we have studied the induction of stem cell factor (SCF) and interleukin-1 (IL-1) by SP in stroma. At 10(-9) mol/L SP, cytokine levels in supernatants were IL-1 alpha, 20 +/- 5 ng/mL; IL-1 beta, 40 +/- 10 ng/mL; and SCF, nondetectable; and the cell-associated levels were SCF, 21 +/- 2 ng/mL; IL-1 alpha, 90 +/- 6 ng/mL; and IL-1 beta, 45 +/- 3 ng/mL. Reverse transcriptase-polymerase chain reaction and ligand-binding studies with stroma stimulated by these two cytokines resulted in (1) NK-1-like receptor mRNA accumulation and (2) downregulation of SP binding sites (day 1) followed by an upregulation (day 3). Low numbers of high-affinity receptors were expressed by day 1 but not by day 3. The results indicate that SP induces IL-1 and SCF in stroma and that these cytokines have the potential to autoregulate NK-R.

Vasoactive Intestinal Peptide-Receptor Imaging for the Localization of Intestinal Adenocarcinomas and Endocrine Tumors

Background Intestinal adenocarcinomas and various endocrine tumors express large numbers of high-affinity receptors for vasoactive intestinal peptide (VIP). We have evaluated the usefulness of scanning with VIP labeled with iodine-123 for tumor localization in patients with gastrointestinal tumors. Methods Radioiodinated VIP was purified by high-pressure liquid chromatography and administered as a single intravenous bolus injection (300 pmol [1 microgram]). Scanning with radiolabeled VIP was compared with computed tomography and scanning with somatostatin analogues in 79 patients with colorectal cancer, pancreatic carcinoma, gastric cancer, carcinoid tumor, or insulinoma. Results Visualization of gastrointestinal tumors and metastases was obtained with radiolabeled VIP. Binding of the labeled peptide by primary tumors and metastases was visible shortly after the injection and was still demonstrable at 24 hours. In patients with colorectal adenocarcinomas, primary or recurrent tumors were visualized in 10 ...