Autocrine growth effect of IL-8 and GROalpha on a human pancreatic cancer cell line, Capan-1.

A human pancreatic cancer cell line, Capan-1, secretes the chemokines interleukin-8 (IL-8) and growth-related oncogene alpha (GROalpha). Capan-1 cells also express the chemokine receptor 2 (CXCR2), which is a Gialpha-protein coupled receptor. Growth of Capan-1 cells was inhibited when anti-IL-8 or anti-GROalpha monoclonal antibody was added into the culture medium. Pertussis toxin, which blocks Gialpha also demonstrated a growth-inhibitory effect on Capan-1 cells. These results indicated that IL-8 and GROalpha act on Capan-1 cells as growth factors in an autocrine manner through CXCR2.

Bronchoalveolar lavage with KL4-surfactant in models of meconium aspiration syndrome.

As a model of the meconium aspiration syndrome (MAS) of human infants, adult rabbits and newborn rhesus monkeys received intratracheal instillation of human meconium to induce pulmonary injury. Injured rabbits were ventilated with 100% O2 and divided into four treatment groups, receiving: 1) bronchoalveolar lavages (BAL) with dilute KL4-Surfactant; 2) lavages with equal volumes of sterile saline; 3) a single intratracheal bolus of KL4-Surfactant, 100 mg/kg; and 4) no treatment. The untreated rabbits developed atelectasis, a fall in pressure-volume levels and in partial pressure of O2 in arterial blood (PaO2) from approximately 500 to < 100 mm Hg, and severe pulmonary inflammation between 3 and 5 h after instillation of meconium. Rabbits treated by BAL with dilute KL4-Surfactant showed rapid and sustained recovery of PaO2 to approximately 300 mm Hg within minutes, a return toward normal pressure-volume levels, and diminished inflammation. Rabbits receiving BAL with saline failed to show recovery, and rabbits treated with a bolus of surfactant intratracheally exhibited a transient response by 1-2 h after treatment, but then returned to the initial atelectatic state. Newborn rhesus monkeys, after receiving human meconium intratracheally before the first breath, developed severe loss of pulmonary function. Treatment of these monkeys 1-5 h after birth with BAL with dilute KL4-Surfactant produced clearing of chest radiographs and a rapid improvement in pulmonary function with ratios of partial pressure of O2 in arterial blood to the fraction of O2 in the inspired air rising into the normal range where they remained through the 20-h period of study. The studies indicate that pulmonary function in two models of severe meconium injury respond rapidly to BAL with dilute KL4-Surfactant.

Importance of the carboxy-terminus of the CXCR2 for signal transduction.

The CXCR2 is phosphorylated at the C-terminal intracytoplasmic portion within 15 sec following the addition of IL-8 or MGSA. Cells transfected with a truncated form of the receptor missing the last 12 amino acids (T3) showed normal binding affinity, but were no longer phosphorylated; individual alanine replacement indicated that Ser346 and 348 were the primary sites of phosphorylation. In studies of the importance of phosphorylation in CXCR2 desensitization, cells expressing wild type CXCR2 lost GTP gamma S binding above basal rate after the first exposure to IL-8, while cells with the T3 mutant retained 60% of their capacity to induce GTP gamma S exchange upon a second exposure to IL-8. In contrast, receptor internalization was not affected by the loss of phosphorylation of the T3 mutant. Further receptor truncation led to decreasing binding affinities for IL-8 and MGSA and a decreased rate of GTP gamma S exchange following addition of excess ligand which suggests involvement of this region in G-protein coupling.

The role of Tyr13 and Lys15 of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A.

Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys7 and Cys50 that, together with the Glu4-Leu5-Arg6 sequence of the NH2 terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule. Replacement of rabbit His13 and Thr15 with Tyr13 and Lys15 of the human molecule converted the low affinity binding of the rabbit IL-8 to the high affinity binding of human IL-8 as shown by both competitive binding and by Ca2+ mobilization. As a corollary, replacement of the Tyr13 and Lys15 of the human IL-8 with His13 and Thr15 of the rabbit IL-8 reduced binding activity of this mutated human IL-8 200-fold. The site of interaction on the IL-8 receptor type A for the Tyr13 and Lys15 sequence was found to be in the NH2-terminal region of this receptor. A structural pattern of the binding between IL-8 and the A type IL-8 receptor is proposed.

Actin polymerization, calcium-transients, and phospholipid metabolism in human neutrophils after stimulation with interleukin-8 and N-formyl peptide.

Signal transduction of interleukin-8 (IL-8) was analyzed in neutrophils, and compared with the well known neutrophil activator N-formyl peptide. Stimulation of human neutrophils with IL-8 induced a rapid polymerization of actin as detected by 7-nitrobenz-2-oxa-1,3-diazol-(NBD)-phallacidin staining of f-actin and reduction of monitored right-angle light scatter. Actin polymerization peaked within 10 seconds after the addition of IL-8 and was short-lived as compared to N-formyl peptide-induced stimulation. Analysis of phospholipids by thin-layer chromatography and analysis of deacylation products of lipid extracts by high-pressure liquid chromatography (HPLC) showed that IL-8 triggered a rapid rise of [32P]phosphatidyl-inositol(3,4,5)trisphosphate (PtdInsP3) followed by a slower increase of [32P]phosphatidylinositol(3,4)bisphosphate (PtdIns-3,4-P2) along with a rapid decrease of [32P]phosphatidylinositol(4,5)bisphosphate (PtdIns-4,5-P2). Changes in polyphosphoinositide metabolism were more moderate and transient than those obtained by N-formyl peptide. Moreover, [32P]phosphatidic acid (PA) production stimulated by IL-8 was minimal and transient as compared to the response activated by N-formyl peptide. Both IL-8 and N-formyl peptide induced Ca++ mobilization from intracellular stores, but IL-8 in contrast to N-formyl peptide failed to trigger the secondary influx of Ca++ from the extracellular medium. In summary, IL-8 and N-formyl peptide stimulated similar and distinct patterns of intracellular activation steps. This study indicates that IL-8 is a potent activator of intracellular events presumably required for chemotaxis, but a relatively weak activator for events associated with superoxide anion generation and proinflammatory activity.

Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors.

To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.

N-formylpeptide-receptor dynamics, cytoskeletal activation, and intracellular calcium response in human neutrophil cytoplasts.

Cytoplasts (enucleated neutrophils which are depleted of dense granules) were prepared from human neutrophils with a modified procedure which employed dihydrocytochalasin B instead of cytochalasin B. These cytoplasts retained an activatable cytoskeletal network similar to cells in that filamentous actin polymerization in response to an N-formylpeptide (fluoresceinated N-formyl-nle-leu-phe-nle-tyr-lys, FLPEP) occurred with similar dose-response characteristics and was inhibitable by cytochalasin B and dihydrocytochalasin B. Cytoplasts had the same number of receptors per surface area as cells and binding constants and dissociation kinetics were the same for cells and cytoplasts. The conversion of receptors from a rapidly dissociating form to a slowly dissociating form was comparable in cells and cytoplasts. This conversion was not inhibited by cytochalasins and thus did not require actin polymerization. Cytoplasts were capable of internalizing 30% of bound FLPEP after 3 min of binding. Cytochalasins did not block this internalization which thus did not appear to require actin polymerization. After 5 min of binding, [3H]-N-formyl-met-leu-phe cosedimented with the Golgi marker enzymes when cytoplasts were fractionated on sucrose density gradients after N2 cavitation. These results indicate that the internalization mechanism is functional in cytoplasts. The Indo-1-detectable calcium response in cytoplasts had a ED50 similar to cells, though the maximum increase in Ca2+ concentration was about one-half that of cells. The response recovered with time after stimulation and the calcium detected was primarily from intracellular stores. The decay of responses after addition of formylpeptide antagonists was parallel for cells and cytoplasts, and leukotriene B4-induced responses in both cells and cytoplasts. Thus the regulation of the responses in cells and cytoplasts was analogous.

Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor.

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.

Signal transduction and ligand-receptor dynamics in the neutrophil. Ca2+ modulation and restoration.

Intracellular Ca2+ rises when neutrophils are stimulated with formyl peptide ligands. There is enough Ca2+ released to complex approximately 200 microM Quin 2, (220 +/- 90 microM, 7 donors). This result is interpreted in terms of a fixed storage pool of Ca2+ of 44 pmol/10(6) cells. When extracellular Ca2+ is removed from the medium with 5 mM EGTA (final pH 7.4) just prior to cell stimulation, neither the magnitude nor the early time course of the Quin 2 response to formyl peptide is dramatically influenced. This result supports the concept that neither Ca2+ influx nor efflux, which are elevated in stimulated cells, contributes in a major way to the free Ca2+ pool which is monitored by Quin 2 during the early activation phase of cell responses. We have used intracellular Quin 2, and extracellular Ca2+ without the use of EGTA or ionophores to manipulate the levels of intracellular Ca2+. This is accomplished by depleting cells of intracellular Ca2+ by loading with Quin 2 in the absence of Ca2+. Intracellular Ca2+ is modulated by adding back Ca2+ to the medium. Using simultaneous analyses of cell function and Quin 2 fluorescence, we find that at least two aspects of cellular responsiveness (degranulation and O2- production) depend upon the level of available Ca2+. In contrast, the first phase, at least, of a biphasic rapid light scattering response which is related to actin polymerization is independent of Ca2+. We find that the Ca2+- sensitive cell responses can be partially restored in Ca2+-depleted cells if Ca2+ is provided within 30 s, a period which may reflect the putative lifetime of the transiently active ligand-receptor complex.

Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation.

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.

The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil.

Parallel cytometric and fluorimetric analyses of the interaction of a fluoresceinated N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, Nle = norleucine) with its receptors on human neutrophils are presented. The cytometric analyses take advantage of the ability of the fluorescence flow cytometer to discriminate free and receptor-bound ligand in a homogeneous real-time assay. The spectrofluorometric analysis relies on a high affinity antibody to fluorescein to discriminate free and bound ligand. We find that the number of receptors for formyl peptides on the surface of a resting cell is 53,000 +/- 13,000 (Kd approximately 0.6 +/- 0.2 nM). We use commercially available cytometric standards to calibrate the cytometer and we obtain similar values for the number of receptors. The temperature dependence of the kinetics of ligand-receptor interactions have been examined. The association rate constant varies from approximately 3 X 10(8) M-1 min-1 at 4 degrees C to approximately 10(9) M-1 min-1 at 37 degrees C (delta H approximately 8 kcal/mol). While ligand internalization is blocked at 4 degrees C, at 37 degrees C internalization proceeds at an initial rate of approximately 24% of the occupied receptors/min following a latency period of approximately 20-30 s. Intermediate rates and longer latency periods are found at 15 and 25 degrees C. Dissociation of the ligand is heterogeneous and depends both on the length of time of association and the temperature. After short periods of association, the ligand dissociates with t1/2 approximately 1-5 min. After longer periods (30 min at 15 degrees C or 100 min at 4 degrees C), but while the ligand-receptor complex remains on the cell surface, t1/2 increases to greater than 30 min. It appears that the ligand-receptor complex undergoes an alteration in affinity, with a time course at elevated temperatures, which parallels or lags behind the time course of the participation of the occupied receptors in cell activation.

Evidence for N-formyl chemotactic peptide-stimulated GTPase activity in human neutrophil homogenates.

Neutrophil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+27%) by the addition of 100 nM N-formyl chemotactic peptide (CHO-pep), but not by 1 microgram X ml-1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 +/- 6.9 s between the addition of CHO-pep and the optimal GTPase stimulation. The GTPase activity (but not CHO-pep-stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO-pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO-pep stimulation of neutrophils is discussed.

Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation.

Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide. When antibody is added to a cell suspension that has been stimulated with the fluoresceinated peptide, we observe that: (i) the apparent membrane depolarization response monitored by a fluorescent dye can be inhibited only if antibody is added within 30 sec of stimulation; (ii) the superoxide response can be inhibited even if antibody is added more than 1 min after stimulation and decays with an intrinsic half-life of about 12 sec; (iii) responses to a second dose of nonfluoresceinated peptide are enhanced if the antibody is added within 2 min of stimulation by the fluoresceinated peptide. These results suggest that different neutrophil responses depend in individual ways on the time course and extent of ligand binding to its receptor. A comparison of these data with the time course of binding permits an estimate of the number of receptors involved in these responses.

A simple and reproducible method to assess murine platelet responses in vitro.

A simple and reproducible technique for murine platelet isolation is described. Such preparations then become useful in determining in vitro platelet secretory responses and platelet aggregation.

Mechanisms of lipopolysaccharide-initiated rabbit platelet responses. II. Evidence that lipid A is responsible for binding of lipopolysaccharide to the platelet.

The mechanism of bacterial lipopolysaccharide-(LPS) initiated, complement-(C) mediated rabbit platelet lysis has been examined. The results of these studies support our previous observations that activation of the alternative C pathway is required for platelet lysis and that preparations of LPS that activate only the classical pathway (e.g., lipid A) do not cause lysis. The temporal relationship of the interaction of the LPS with the platelet before the addition of plasma suggests a time-dependent association of the LPS with the platelet. On the basis of a number of experiments, including inhibition with polymyxin B, treatment of LPS with alkali, and blocking experiments with polysaccharide-free LPS preparations, it is concluded that the lipid A region of the LPS molecule is responsible for attaching the LPS to the platelet. Finally, a comparison of the activity of lipid A-associated protein-LPS complexes with protein-free LPS demonstrated that an equivalent extent of platelet lysis was achieved with one-one hundredth the concentration of the former as that required for protein-free LPS. The data suggest that LAP facilitates attachment of the LPS to the platelet.

Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: alternative complement pathway dependence of the lytic response.

Experiments were performed to examine the relationship of endotoxin-initiated complement activation and rabbit platelet lysis. The results of these experiments supported the concept that activation of the alternative pathway is required for endotoxin-initiated complement-dependent rabbit platelet lysis. Our data demonstrated that preparations of endotoxin or isolated lipid A, which activate selectively the classical pathway, are incapable of initiating platelet lysis. Essentially equivalent results were obtained in citrated or heparinized plasma, although the latter anticoagulated plasma appeared to be more efficient in supporting lysis. Additional data support the concept that natural antibody to either the polysaccharide or the lipid A region of the lipopolysaccharide, which might be present in rabbit plasma, probably did not play a prominent role in the complement-mediated lytic response.

Activation of platelets by platelet-activating factor (PAF) derived from IgE-sensitized basophils. II. The role of serine proteases, cyclic nucleotides, and contractile elements in PAF-induced secretion.

Secretion of serotonin from platelets induced by platelet-activating factor (PAF) derived from antigen-stimulated, IgE-sensitized rabbit basophils was studied to further characterize the biochemical requirements. Inhibition of secretion with diisopropylphosphofluoridate (DFP) was observed if the DFP was present during the reaction, but not if platelets or PAF were pretreated with the inhibitor. This suggested a role for an activatable serine protease in the secretion. Supporting evidence came from the observation that other protease inhibitors and a variety of low molecular weight amino acid esters were also inhibitory. TAMe was most effective, and AGLMe and LeuMe were inactive, indicating a specificity for different esters. Secretion was reduced by agents that increased intracellular cyclic AMP (cAMP), but enhanced by alpha-adrenergic stimulation, which reduced the levels of cAMP. Concurrent with PAF-induced secretion, a reduction in cAMP levels was observed. No effect of cyclic GMP or cholinergic stimulation was found. Secretion was inhibited by colchicine and enhanced by cytochalasin B, suggesting a role for microfilaments and microtubules. The effects of these three systems on PAF-induced secretion indicate the basic uniformity of the secretory process in platelets (and other cells) whatever the stimulus. The uniqueness of the reaction apparently lies in the stimulus-receptor interaction and the nature of the serine protease which is activated.

Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Secretion of granule constituents and increased oxidation of glucose.

Reaction of human neutrophils with aggregated immunoglobulin on nonphagocytosable surfaces results in secretion of granule enzymes (exocytosis of granules) and stimulation of glucose oxidation by the nexose monophosphate pathway (HMP). The role of HMP stimulation in the enzyme secretion and some requirements for the two neutrophil activities have been examined. It was found that (a) HMP stimulation could be selectively inhibited under conditions where release of granule enzymes remained unchanged or was enhanced, for example, by reduced glucose concentration or by 2-deoxyglucose. (b) Removal of Ca++ and addition of agents which increased the intracellular levels of cyclic AMP (cAMP), however, prevented both activities, while colchicine had greater inhibitory activity on HMP stimulation than upon secretion. (c) Neutrophils incubated in suspension with particulate aggregated gamma-globulin phagocytosed the particles and exhibited a stimulated HMP and released granule enzymes. In contrast, incubation in suspension with soluble aggregated gamma-globulin resulted in the stimulated HMP only. Granule evzymes were not liberated. 300-fold less soluble aggregates bound to a surface, however readily induced exocytosis of granules from adherent neutrophils. This demonstrates the importance of surface effects in the induction of secretion from neutrophils. Aggregated immunoglobulin reacting with neutrophil Fc receptors thus induces both degranulation (exocytosis) and increased HMP activity. The pathways leading to these events are separable although apparently sharing some common steps, including the initiating events.