Enhanced neutrophil respiratory burst as a biological marker for manipulation forces: duration of the effect and association with substance P and tumor necrosis factor.

A critical need in assessing the clinical utility of manipulative therapy for back pain is the identification of biological changes associated with the forces applied by spinal manipulation. Such changes could then serve as markers for both sham treatment and manipulation. We determined the priming of polymorphonuclear neutrophils for an enhanced respiratory burst and its duration, the priming of mononuclear cells for enhanced endotoxin-stimulated tumor necrosis factor production and plasma levels of substance P following a single thoracic spine manipulation. There was a significant difference in the respiratory burst of polymorphonuclear neutrophils in response to a particulate challenge, depending on the time of blood sample collection. The response of polymorphonuclear neutrophils isolated from blood collected 15 min after manipulation was significantly higher than the response of cells isolated from blood collected 15 min before and 30 and 45 min after manipulation. Mononuclear cells were also primed for enhanced endotoxin-stimulated tumor necrosis factor production by spinal manipulation. Both of these priming effects were accompanied by a slight, but significant elevation in plasma substance P. The mean manipulation force associated with these biological effects was 878 +/- 99 N. These biological effects may provide a means of monitoring the delivery of both sham and manipulative treatment and, therefore, provide a crucial tool for understanding the efficacy of manipulative therapy.     

Impairment of polymorphonuclear leukocyte function and metabolic control of diabetes.

OBJECTIVE--In this study, ingestion of Staphylococcus aureus and "bacteria killing" (BK) were measured to evaluate polymorphonuclear leukocyte (PMN) phagocytic functions and chemiluminescence response (CL) to phorbol-myristic acetate (PMA) as respiratory burst activity with regard to metabolic control parameters in diabetic patients. RESEARCH DESIGN AND METHODS--PMN phagocytic functions were assessed in 40 diabetic patients, all receiving insulin and in poor metabolic control, with 3H-thymidine-labeled Staphylococcus aureus in a modified radiometric assay. Bacteria killing was determined by pure-plate counting of surviving bacteria (colony-forming units [cfu]) and luminol-enhanced CL in response to PMA as a measure of respiratory burst. PMN function data were correlated to HbA1 as parameter of recent metabolic control. RESULTS--PMN of diabetic patients showed a significant reduction in Staphylococcus aureus (50.7 +/- 4.1%) and BK (29.4 +/- 4.2%) compared with healthy nondiabetic control subjects (76.6 +/- 4.6% and 16.3 +/- 3.1%, respectively, P less than 0.001), and PMN CL response was markedly reduced in diabetic patients also. Linear regression analysis showed a highly significant negative correlation of HbA1 versus Staphylococcus aureus (r = -0.67, P = 0.001) and a positive correlation for BK (r = 0.73, P less than 0.001). This was also true for CL, although this did not reach statistical significance (P = 0.06). CONCLUSIONS--The data obtained demonstrate impaired PMN phagocytic functions and CL response in diabetic patients. These findings suggest inhibitory effects of elevated glucose concentrations on PMNs, a possible role of protein glycosylation for impairing PMN function, thus contributing in part to altered host defense.     

Inflammatory response of neutrophil granulocytes and monocytes after cardiopulmonary bypass in pediatric cardiac surgery

OBJECTIVE: To determine whether the activation state of polymorphonuclear neutrophils (PMNs) and monocytes contributes to the inflammatory response after cardiopulmonary bypass (CPB) in pediatric cardiac surgery. DESIGN: Observational prospective clinical study. SETTING: Pediatric intensive care unit of a university hospital. PATIENTS: Twenty pediatric patients before and after open heart surgery with CPB. MEASUREMENTS: Cell counts of circulating PMNs and monocytes as well as phenotypic and functional analysis of these cells, and plasma levels of myeloperoxidase. RESULTS: Levels of myeloperoxidase (a marker of PMN degranulation) were significantly elevated after CPB (2.9+/-1.6 ng/ml before CPB versus 13.7+/-6.5 ng/ml after CPB, p=0.0001). However, PMN function, as measured by surface expression of CD11b/CD18 and phagocytic respiratory burst, was reduced. In contrast, the phagocytic respiratory burst of circulating monocytes was increased in some patients and there was a correlation with the increase of monocyte cell count after CPB (r=0.63, p=0.015). CONCLUSIONS: After the end of CPB, there was an ongoing inflammatory process. In particular, there was an activation of monocytes after the end of CPB.     

The effect of spinal manipulation on pain and prostaglandin levels in women with primary dysmenorrhea.

OBJECTIVE: The primary objectives of this study were to compare the effect of spinal manipulation vs. sham manipulation on a) circulating plasma levels of the prostaglandin F2a metabolite, 15-keto-13,14-dihydroprostaglandin (KDPGF2a), b) perceived abdominal and back pain and c) perceived menstrual distress in women with primary dysmenorrhea. DESIGN: This randomized clinical pilot study investigated the outcome measures before and after either a spinal manipulation treatment (SMT) or a sham manipulation. SETTING: All subjects were treated at the National College Chiropractic clinic, a private chiropractic clinic in the suburban Chicago area. PARTICIPANTS: Forty-five women with a history of primary dysmenorrhea were recruited from the local community. The volunteers ranged in age from 20-49 (mean age = 30.3 yr), and were entered into the study between April 1990 and January 1991. Twenty-four were randomly assigned to the spinal manipulation group and 21 were assigned to the sham group. INTERVENTIONS: Subjects treated with spinal manipulation were placed in a side-lying position with the bottom leg straight and the top leg flexed at the knee and hip. They received a high-velocity, short lever, low-amplitude thrust to all clinically relevant vertebral levels within T10 and L5-S1 and the sacroiliac joints. In the sham manipulation, subjects were placed in a side-lying position with both hips and knees flexed. Their manipulation consisted of a similar thrust administered to the midline base of the sacrum. OUTCOME MEASURES: Perceived abdominal and back pain were measured with a visual analog scale, and menstrual distress was measured with the Menstrual Distress Questionnaire. Both were administered 15 min before and 60 min after treatment. Blood samples were collected by venipuncture for the determination of plasma levels of KDPGF2a at the same times. The plasma was then assayed for KDPGF2a by radioimmunoassay. RESULTS: Analysis of covariance and paired Student's t tests were used for the statistical evaluation. Immediately after treatment, the perception of pain and the level of menstrual distress were significantly reduced by SMT. This reduction was associated with a significant reduction in plasma levels of KDGPF2a in the SMT group. A significant and similar reduction in plasma KDPGF2a also occurred in the sham group, indicating that a placebo effect was associated with a single sham intervention. CONCLUSIONS: This randomized pilot study suggests that SMT may be an effective and safe nonpharmacological alternative for relieving the pain and distress of primary dysmenorrhea. However, the large change in KDPGF2a observed in both treatment groups clearly indicates that further studies with more subjects, studied over a longer time frame, are needed to resolve the question of a placebo effect.     

Identification and characterization of a monocyte-derived neutrophil-activating factor in corticosteroid-resistant bronchial asthma

Peripheral blood mononuclear cells (PBMC) were isolated from seven normal subjects, eight asthmatic subjects clinically sensitive to corticosteroids (CS), and eight asthmatic subjects clinically resistant to corticosteroids (CR). PBMC were cultured at 37 degrees C for 24 h in the absence or presence of 10(-16) to 10(-4) M hydrocortisone. Calcium ionophore (A23187)-activated neutrophils (PMN) primed by supernatants of PBMC from asthmatic subjects cultured in the absence of hydrocortisone generated approximately threefold more leukotriene B4 than PMN primed by supernatants of PBMC from normal subjects (P less than 0.05). Incubation of PBMC derived from CS subjects with 10(-8) M hydrocortisone completely inhibited the production of the enhancing activity (P less than 0.01), whereas in CR subjects hydrocortisone at concentrations up to 10(-4) M did not suppress the release of enhancing activity. The enhancing activity was produced by monocytes. Enhancing activity eluted with an Mr of 3,000 D and a pI of 7.1. It eluted at 10% acetonitrile after reverse-phase HPLC. The activity was destroyed by heating to 60 degrees C for 60 min and was sensitive to pronase treatment. The purified factor also enhanced superoxide generation by PMN which had been stimulated submaximally by phorbol myristate acetate.     

Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes.

Recombinant tumor necrosis factor alpha (rTNF alpha) and beta (rTNF beta) did not trigger H2O2 release from PMN in suspension. However, when PMN were plated on polystyrene surfaces coated with serum, fibronectin, vitronectin, laminin, or human umbilical vein endothelial cells (HUVEC), rTNFs induced a massive, prolonged secretory response, similar to that elicited by phorbol myristate acetate (PMA) or bacteria. On serum-coated plates, the maximum sustained rate of H2O2 release in response to rTNF alpha was 2.6 +/- 0.2 nmol/min per 10(6) PMN, the same as that with PMA; release continued for 73 +/- 4 min. On laminin-coated surfaces or HUVEC, release of H2O2 in response to rTNFs was slower, but lasted approximately 3.5 h, reaching the same total (greater than 100 nmol/10(6) PMN). Not only was this response far longer and larger than for other soluble stimuli of the respiratory burst studied with PMN in suspension, but the concentration necessary to elicit a half-maximal response (EC50) for rTNF alpha was orders of magnitude lower (55 pM). Responses were similar with FMLP, but ranged from zero to small with recombinant IFN alpha, recombinant IFN beta, recombinant IFN gamma, platelet-derived growth factor, recombinant IL-1 beta, or bacterial lipopolysaccharide. Adherent monocytes did not secrete H2O2 in response to rTNFs. H2O2 secretion by adherent PMN was first detectable 15-90 min after addition of rTNFs or FMLP. This lag period was unaffected by prior exposure of PMN to rTNF alpha in suspension, by allowing PMN to adhere before adding rTNF alpha, or by incubating adherent PMN in medium conditioned by rTNF alpha-treated PMN. Cytochalasins abolished H2O2 secretion in response to rTNFs, but not FMLP, if added during, but not after, the lag period. Thus, H2O2 secretion from rTNF alpha-treated PMN appears to be a direct but delayed response that requires assembly of microfilaments during exposure to the cytokine. These results suggest that PMN adherent to intra- or extravascular surfaces may undergo a massive, prolonged respiratory burst at the command of macrophages and lymphocytes reacting to microbial products and antigens.     

Entry of gut lymph into the circulation primes rat neutrophil respiratory burst in hemorrhagic shock.

OBJECTIVE: Endothelial cell injury by polymorphonuclear neutrophil (neutrophil [PMN]) respiratory burst after trauma and hemorrhagic shock (T/HS) predisposes subjects to acute respiratory distress syndrome and multiple organ failure. T/HS mesenteric lymph injures endothelial cell and lymph duct ligation (LDL) before T/HS prevents pulmonary injury. We investigated the role of mesenteric lymph in PMN priming by T/HS. DESIGN: Prospective experiment in rats. SETTING: University hospital laboratory. SUBJECTS: Adult male rats. INTERVENTIONS: Mesenteric lymph was obtained from rats undergoing T/HS (30 mm Hg, 90 mins) or sham shock (T/SS). Plasma was harvested from uninstrumented control (UC), T/HS, T/SS, and T/HS+LDL rats. PMNs were isolated from UC, T/HS, and T/HS+LDL rats. MEASUREMENTS AND MAIN RESULTS: PMNs from UC rats were incubated in buffer, 1% T/HS lymph, and 1% T/SS lymph. PMNs from UC rats were incubated in UC, T/HS, T/SS, and T/HS+LDL plasma. PMN respiratory burst was initiated by using macrophage inflammatory protein (MIP)-2/platelet-aggregating factor (PAF) or phorbol myristate acetate. Cytosolic calcium ([Ca2+]i) responses to MIP-2/PAF were assayed in PMN from UC, T/HS, and T/HS+LDL rats. PMN preincubated in T/HS lymph showed significant elevations in MIP/PAF-elicited respiratory burst compared with T/HS lymph or buffer only (p <.05; analysis of variance/Tukey's test). T/HS lymph incubation also increased (p <.05) phorbol myristate acetate elicited respiratory burst compared with buffer or T/SS. Preincubation in T/HS plasma increased MIP-2/PAF-elicited respiratory burst (p <.05) compared with UC or T/SS plasma. LDL blocked T/HS priming of respiratory burst. Control PMN [Ca2+]i responses to MIP-2 and PAF were low. T/SS PMN were significantly more responsive, but the T/HS PMN showed still higher responses (p <.01). LDL reversed the priming of [Ca2+]i responses by T/HS (p <.01). CONCLUSIONS: PMNs are primed by T/HS lymph but not T/SS lymph and by T/HS plasma but not T/SS plasma. LDL before shock prevents T/HS plasma from priming PMN. The magnitude of respiratory burst found here paralleled the [Ca2+]i responses seen to receptor dependent initiating agonists. Mesenteric lymph is both necessary and sufficient to prime PMN after T/HS in the rat, and it primes PMN in part by enhancing [Ca2+]i responses to G-protein coupled chemoattractants. Mesenteric lymph mediates postshock PMN dysfunction.     

Post-hemorrhagic shock mesenteric lymph lipids prime neutrophils for enhanced cytotoxicity via phospholipase A2.

Hemorrhagic shock induced mesenteric hypoperfusion has long been implicated as a key event in the pathogenesis of the adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Previous work links post-hemorrhagic shock mesenteric lymph (PHSML) lipids and neutrophil (PMN) priming in the pathogenesis of ARDS. We hypothesize that gut phospholipase A2 (PLA2) liberates proinflammatory lipids following hemorrhagic shock, which are responsible for enhanced PMN cytotoxicity. Mesenteric lymph was collected from rats (n > or = 5) before hemorrhagic shock, during hemorrhagic shock (MAP 40 mm Hg x 30 min), and after resuscitation (shed blood + 2x lactated Ringers). PMNs were incubated with physiologic concentrations (1-5%, v:v) of (a) buffer control, (b) sham (c) pre-shock lymph, (c) PHSML, (d) PHSML lipid extracts, (e) heat-denatured PSHML, and (f) PHSML harvested after i.v. pretreatment with a known PLA2 inhibitor (quinacrine, 10 mg/kg). PMNs were activated with fMLP (1 micromol), and the maximal rate of superoxide production measured by reduction of cytochrome c. Gut morphology was assessed histologically using hematoxalin and eosin (HE) staining. PHSML and PHSML lipid extracts (5%, v:v) primed for enhanced superoxide production compared to buffer controls (2.5-fold and 3.6-fold), sham (2.5-fold) and pre-shock lymph (2.0-fold). Lymph collected after systemic PLA2 inhibition, in contrast, abrogated the PMN priming response. Gut mucosal morphology, at end-resuscitation, was intact on HE staining both with and without PLA2 inhibition. Heat denaturing the PHSML (eliminating cytokines and complement), on the other hand, did not reduce PMN priming. Physiologic concentrations of PHSML lipids prime the PMN respiratory burst. Lymph priming is diminished with systemic PLA2 inhibition, implicating gut PLA2 as a source of proinflammatory lipids that may be central in the pathogenesis of hemorrhagic shock induced ARDS/MOF.     

Somatovisceral response following osteopathic HVLAT: a pilot study on the effect of unilateral lumbosacral high-velocity low-amplitude thrust technique on the cutaneous blood flow in the lower limb

INTRODUCTION: Spinal manipulative treatment is widely used among manual therapists, although knowledge regarding the absolute physiological effects has not been clearly established. In this study, 20 healthy male subjects underwent a unilateral high-velocity low-amplitude thrust (HVLAT) to the lumbosacral junction, while the cutaneous blood flow in the corresponding dermatome of the lower limb was monitored. METHODS: Subjects underwent a sham manipulation before the actual manipulation and acted as their own control. Laser Doppler flowmetry was used to measure relative changes in the cutaneous blood flow over the L5 dermatome for 5 minutes before the sham manipulation, for 5 minutes between the sham and the actual manipulation, and for 5 minutes after the spinal adjustment. Analysis of variance (ANOVA) and Tukey post hoc analysis was used in the interpretation of the data. RESULTS: Twelve nonsmoking subjects, who received a successful HVLAT manipulation, showed a significant increase (P <.001) in blood perfusion, both ipsilaterally and contralaterally. Six smokers responded with a significant decrease in blood flow ipsilaterally (P <.01) and contralaterally (P <.001) after HVLAT manipulation. CONCLUSION: The results from this study support previous published hypotheses that spinal adjustments outside the region of the sympathetic outflow result in an increase in cutaneous blood flow. Further studies will be needed to confirm the outcome of this study, and more knowledge is needed regarding the specific neurophysiological effects of spinal manipulation.     

Re-evaluation of the phagocytic respiratory burst in the physiological or inflammatory state and in aging

We studied the differences in oxygen metabolite generation, using a chemiluminescence (CL) assay, in peripheral blood phagocytic cells from various donors including healthy young volunteers, patients with acute or chronic inflammation, pregnant women, and elderly persons. The CL response of polymorphonuclear leukocytes (PMN) after stimulation with serum-opsonized zymosan was increased in patients with acute inflammation due to infection and in pregnant women as compared with that in controls. Monocytes from those patients also showed a slight increase of the CL response. In contrast, CL of monocytes from patients with chronic inflammation (Crohn's disease patients) and elderly persons was significantly enhanced, whereas that of their PMN remained in the range of control values. The significance of these results was discussed.     

Spinal manipulation and beta-endorphin: a controlled study of the effect of a spinal manipulation on plasma beta-endorphin levels in normal males

The role of spinal manipulation in the relief of pain is becoming clearer and more demonstrable as time passes. One approach to this study is the effect of manipulation on the neurochemical mechanisms of antinociception. Chief among these is beta-endorphin, which has been found to produce a wide range of beneficial effects, especially analgesia. The intent of our study was to demonstrate the effect of spinal manipulation on plasma beta-endorphin levels. Three groups of male subjects were randomly created: the experimental, sham and control groups. All three groups were screened for symptomatology, present use of medications and the present use of innocuous stimulants, such as nicotine and caffeine. A standard protocol involving a 20-min pretest resting period, an intervention and a 40-min test period ensued. The experimental group received a manipulation in the region of the cervical spine; the placebo group received a sham maneuver with no dynamic thrust; the control group received no intervention. Samples were taken by venipuncture at -20, -5, +5, +10 and +30 min. The data were analyzed by repeated measures analysis of variance and by Scheffe's post-hoc multiple comparison tests. Plasma beta-endorphin levels were assessed by radioimmune assay technique (according to the method described by Harber and Sutton in 1984). The results of our study demonstrated a small, but statistically significant, increase in serum beta-endorphin levels in the experimental group at the 5-min postintervention point. The levels in the placebo and control groups demonstrated a steady decrease that was distinct from the response in the experimental group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonsteroidal anti-inflammatory drug as tools for analysis of neutrophil functions

We investigated the effects of the nonsteroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone on the activities relating to the migration and respiratory burst of polymorphonuclear leukocytes (PMN). When diclofenac sodium, was incorporated into the agarose gel at various concentrations below 100 micrograms/ml, it inhibited, in a dose-dependent fashion, spontaneous PMN migration and the directional migrations induced by both C5a-activated serum and peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). By contrast, phenylbutazone (below 100 micrograms/ml) only altered the directed PMN migration induced by FMLP, in two characteristic ways: by impairing the optimal response to 10(-7) M FMLP, and in particular, by restoring the loss of migration induced by higher but deactivating concentrations of 10(-6) and 10(-5) M. Indomethacin had similar effects to those of phenylbutazone on FMLP-induced PMN migration and in addition slightly impaired spontaneous PMN migration. The alterations in FMLP-induced migration caused by the three drugs tested were mainly chemokinetic and were due to changes in migratory speed. Of the three drugs, phenylbutazone and indomethacin also impaired FMLP-induced changes in the shape of PMN. All three interfered with the respiratory burst induced by FMLP but not with that induced by phorbol myristate acetate. These results demonstrate that phenylbutazone, indomethacin and diclofenac possess different spectra of biological activities as regards the parameters relating to PMN migration and respiratory burst, and therefore suggest that these drugs could serve as tools for investigating PMN functions.     

Membrane-bound lactoferrin alters the surface properties of polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMN) aggregate and avidly attach to endothelium in response to chemotactic agents. This response may be related in part to the release of the specific granule constituent lactoferrin (LF). We found by using immunohistology and biochemical and biophysical techniques that LF binds to the membrane and alters the surface properties of the PMN. Upon exposure of PMN treated with 5 micrograms/ml cytochalasin B to 2 x 10(-7) M formyl-methionine-leucine-phenylalanine for 5 min, the PMN mobilized LF to their surface as observed by immunoperoxidase staining for LF. At added LF levels ranging from 4 to 15 micrograms/10(7) PMN there was a dose-dependent reduction in PMN surface charge reaching 4 mV, when the partitioning into the membrane of a charged amphipathic nitroxide spin label was measured by electron spin resonance spectroscopy, whereas transferrin was without effect. When 125I-FeLF was added to human PMN in increasing amounts and the results corrected for the residual amount of free LF contaminating the cells, the PMN were saturated with LF at concentrations between 100 and 200 nM in the medium. Human PMN bound 1.35 x 10(6) molecules per cell and the calculated value for the association constant for these receptors was 5.2 x 10(6) M-1. Additionally, 6 micrograms/ml LF served as an opsonin for rabbit MN to promote PMN uptake by rabbit macrophages, when assessed by electron microscopy, but lysozyme did not. These studies indicate that LF can bind to the surface of the PMN and reduce its surface charge. This correlates with enhanced "stickiness" leading to a variety of cell-cell interactions.     

Adhesion receptors of polymorphonuclear granulocytes on titanium in contact with whole blood

Titanium sheets were exposed to whole blood, and the TiO(2) surface was investigated regarding the presence of cells, receptor expression on adherent polymorphonuclear (PMN) granulocytes, and the ability of these cells to mount a respiratory burst when challenged with opsonized zymosan. The techniques used were immunofluorescence with computer-aided image analysis and chemiluminescence. Surface coverage of erythrocytes (9% to 10%), granulocytes (9% to 14%), and platelets (1% to 4%) dominated during the first 2 hours of blood contact. PMN granulocyte adhesion to titanium was associated with a rapid decrease in L-selectin expression within 16 minutes. Initially FcgammaIII receptor (CD16) expression dominated on the adherent cells. After 30 minutes, a shift toward integrin expression (CD11b) was found on the adherent cells. All investigated receptors were down-regulated within 1 hour of blood-titanium contact. Attempts were made to inhibit the initial adhesion of PMN granulocytes to titanium by adding specific antibodies or 2,3-diphosphoglyceric acid (phospholipase D inhibitor) to blood before surface contact. Adding anti-CD16 resulted in a 67% reduction in cell adhesion, whereas a 35% reduction was found with 2,3-diphosphoglyceric acid. No spontaneous respiratory burst was detected from adherent PMN granulocytes residing on the TiO(2) surface. The cells were, however, able to mount a respiratory burst in response to opsonized zymosan.     

Effects of low concentrations of arachidonic acid derived mediators on the membrane potential and respiratory burst responses of human neutrophils as assessed by flow cytometry

Hypaque-Ficoll purified (95%) neutrophils (PMN) from normal healthy subjects were assessed for FMLP-elicited membrane potential (delta psi) responses and dichlorofluorescein (DCF) fluorescence (a measure of intracellular hydrogen peroxide production) using flow cytometry and appropriate fluorescent probes. Superoxide (O2) production was measured spectrophotometrically as the superoxide dismutase-inhibitable reduction of cytochrome C. The modulatory effects of dilute solutions of the arachidonic acid-derived inflammatory mediators LTB4, LTC4, LTD4, PGE1, PGE2 and PGF2 alpha (10(-9)-10(-5) M) were assessed in these systems. While LTB4 enhanced the proportion of cells depolarizing to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) 2-3x with a maximum effect in the 10(-9)-10(-8) M range, LTC4 and LTD4 showed no such enhancement except at high concentrations (10(-6) M). Unlike LTB4, LTC4 and LTD4 were unable to enhance FMLP mediated PMN O2 or DCF responses at any concentration tested, implying a divergence between the effects of the leukotrienes on membrane potential and oxidant responses. Pre-incubation of PMN with prostaglandins E1 or E2 led to a dose dependent inhibition of the proportion of depolarizing PMN in response to FMLP; PGF2 alpha did not show such an effect. The present data indicate that LTB4, in addition to being a powerful direct neutrophil activator, may act in a priming capacity by increasing the proportion of subsequently FMLP responsive cells, while PGE's inhibit. These modulatory effects appear relatively specific for LTB4 and the E-series prostaglandins.     

Monocyte functional and metabolic activity in malignant and inflammatory diseases.

Macrophages or monocytes produce CL upon exposure to ingestible particles such as opsonized zymosan or bacteria. In previous studies, we have demonstrated that activated macrophages from mice produce significantly more CL than do normal macrophages. In the present study, we have utilized the CL assay as well as 14C-1-glucose utilization to assess monocyte metabolic activity in a variety of malignant, infectious, and inflammatory diseases. Monocyte peak CL was significantly increased above control values (20.9 +/- 0.5 (S.E.) X 10(3) cpm) in 25 patients with lymphoma (26.7 +/- 1.5 x 10(3)). Markedly increased CL was also seen in inflammatory processes such as bacterial infections, tuberculosis, and sarcoidosis (32.2 +/- 2.7 x 10(3)). In contrast, monocytes from patients with solid tumors, including carcinomas of breast and gastrointestinal and genitourinary tracts, had peak CL values (22.4 +/- 1.6 x 10(3) which were not significantly different from controls. When studied by determining 14C-1-glucose utilization, hexose monophosphate shunt activity paralleled CL values. Monocyte metabolic activation appears therefore to accompany ongoing infectious or granulomatous processes and may also be present in certain malignancies associated with reticuloendothelial stimulation.     

Vasopressin, aldosterone and renin responses to volume depletion in heart-transplant recipients

Plasma arginine vasopressin (AVP), renin activity (PRA) and aldosterone (ALD) were measured immediately before and 60 min after intravenous administration of frusemide and passage from lying to standing in 10 untreated healthy subjects (group 1), eight asthmatic patients treated with prednisone (group 2) and 13 heart-transplant recipients treated with prednisone and cyclosporin (group 3). Three different tests for cardiac vagal innervation were performed in the study population. They confirmed that the patients of group 3 were denervated whereas those of groups 1 and 2 had an intact cardiac innervation. Plasma volume depletion after frusemide administration estimated from the rise in plasma proteins was 10-12%. Mean blood pressure was higher in the transplant recipients but did not change in the three groups. Heart rate was also greater in the transplant recipients as a result of vagal denervation. PRA and ALD increased in all the subjects: 2.8, 3.3 and 2.2 times basal value for PRA, 2.7, 4.6 and 2.1 times basal value for ALD in groups 1, 2 and 3 respectively. In contrast, plasma AVP increased only in the two control groups (x1.45 and x1.65 in groups 1 and 2 respectively) whereas it was unchanged in the group of heart-transplant recipients (x1.05). In order to better understand the etiology of the high basal AVP plasma levels observed in group 3, AVP response to a standard water load was studied in eight supplementary heart-transplant recipients: 81.5% of the water load was excreted over 3 h and plasma AVP fell significantly (x0.76).(ABSTRACT TRUNCATED AT 250 WORDS)     

Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin.

Entactin is an integral component of basement membranes that plays a major role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Because neutrophil (PMN) interactions with entactin have not been examined, we investigated the ability of natural and recombinant entactin to mediate PMN adhesion and chemotaxis. With both forms of entactin, we observed that entactin-coated surfaces promoted PMN adhesion and that entactin stimulated PMN chemotaxis. The increase in adhesion to entactin over control was two to threefold whereas the chemotactic response to 15 ng/ml (1 x 10(-10) M) entactin was equivalent to the chemotactic response elicited with 1 x 10(-8) M formyl-methionyl-leucyl-phenylalanine (fMLP). HL-60 cells, after differentiation with dimethylsulfoxide, also demonstrated adhesion and chemotaxis to entactin. A synthetic peptide of the Arg-Gly-Asp (RGD) domain in entactin, SIGFRGDGQTC (S-RGD), mediated PMN adhesion and chemotaxis, and preexposure of PMN to S-RGD blocked PMN adhesion and chemotaxis induced by entactin without diminishing the adhesive and chemotactic activities of fMLP. In contrast, preexposure to peptides SIGFRGEGQTCA or SIGFKGDGQTCA had no effect. The findings with synthetic peptides were confirmed with a recombinant entactin mutant in which aspartic acid at residue 674 was replaced with glutamic acid, thus converting the RGD sequence of entactin to RGE. RGE-entactin was neither adhesive nor chemotactic for neutrophils. Monoclonal antibodies to the leukocyte response integrin (LRI) and the integrin-associated protein blocked entactin-mediated adhesion and chemotaxis whereas monoclonal antibodies to beta 1 and beta 2 integrins had no effect and PMN from an individual with leukocyte-adhesion deficiency adhered normally to entactin-coated surfaces. These data demonstrate that entactin mediates biologically and pathologically important functions of PMN through its RGD domain and that LRI, which has been shown previously to mediate RGD-stimulated phagocytosis, is also capable of mediating RGD-stimulated PMN adhesion and chemotaxis.     

Diminished Polymorphonuclear Leukocyte Adherence: FUNCTION DEPENDENT ON RELEASE OF CYCLIC AMP BY ENDOTHELIAL CELLS AFTER STIMULATION OF β-RECEPTORS BY EPINEPHRINE

To investigate the biochemical and cellular basis for the rise in polymorphonuclear leukocyte (PMN) count during epinephrine administration, PMN from subjects receiving epinephrine were studied for their capacity to adhere to nylon wool fibers and endothelial cell monolayers. After administration of epinephrine, the PMN count increased by 80% at 5 min, and isolated PMN adherence to nylon fibers fell from a base line of 44±2-18±3%. In contrast, when subjects were infused with the β-antagonist propanolol before receiving epinephrine, the PMN count failed to rise and PMN adherence was normal. Exposure of PMN endothelial cell monolayers to 0.1 μM epinephrine led to diminished PMN adherence that could be blocked by 10 μM propanolol but not by 10 μM phentolamine. Sera obtained from subjects 5 min after receiving epinephrine or from supernates derived from endothelial cell monolayers exposed to 90 nM epinephrine inhibited PMN adherence to nylon fibers. Addition of anticyclic AMP antisera but not anticyclic guanosine monophosphate antisera to the postepinephrine sera or to the postepinephrine supernate derived from the endothelial cell monolayers abolished their inhibitory effect of PMN adherence to nylon fibers. In contrast, direct exposure of PMN to epinephrine failed to affect their adherent properties. Because it has been previously shown that endothelial cells contain β-receptors and respond to catecholamines by raising their intracellular concentrations of cyclic AMP, and that PMN adherence is attenuated by cyclic AMP, it would appear that diminished PMN adherence after epinephrine administration is mediated through endothelial cell β-receptor activity, which in turn impairs PMN margination in vivo and could account for the rise in circulating PMN.