Role of the antibody in the pathogenesis of transplant vascular sclerosis: a hypothesis.
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Animals↗
Biomedical subjects
M D Wewers
Publications and source records attributed to M D Wewers.
Changes in mononuclear phagocyte microtubules after endotoxin stimulation. I. Changes in microtubule stability.
Microtubules are in a dynamic equilibrium of polymerization and depolymerization. In monocytes and macrophages, microtubules bind endotoxin and partly regulate inflammatory events such as cytokine production. To characterize the morphologic differences between alveolar macrophage and blood monocyte microtubules after LPS stimulation, cells were examined by immunofluorescent microscopy and laser confocal microscopy. Fresh monocytes contained an average of 26 microtubules per cell which significantly increased to 31 microtubules per cell following a 30-min exposure to LPS (P < 0.001). Using a nocodazole-based assay of microtubule dynamic instability, the half-life of fresh unstimulated human monocyte microtubules was approximately 18 s and extended to 26 s following a 30-min exposure to LPS. In vitro maturation of monocytes for 18 h increased microtubule stability but not number. Compared to monocytes, alveolar macrophage microtubules were longer, more numerous, and much more stable. These results suggest that alveolar macrophage microtubules are more numerous and stable than blood monocyte microtubules and that LPS causes an increase in monocyte microtubule number and stability.
Female↗
Changes in mononuclear phagocyte microtubules after endotoxin stimulation. II. Changes in microtubule composition.
Microtubules are integral components of the cytoskeleton of human cells and are composed of alpha- and beta-tubulin as well as a variable number of microtubule-associated proteins. In monocytes and macrophages, microtubules bind endotoxin and partly regulate endotoxin-induced inflammatory events such as cytokine production. Endotoxin causes a rapid alteration in monocyte microtubule stability. To characterize the effect of endotoxin on mononuclear phagocyte microtubule composition, Western blots and flow cytometry were performed on human monocytes and the monocyte/macrophage-like cell line THP-1. Compared to unstimulated monocytes, monocytes stimulated with endotoxin for 18 h had increased quantities of alpha-, beta-, and tyrosinated alpha-tubulin as well as microtubule-associated protein-2. PMA-differentiated THP-1 cells had increased levels of alpha-tubulin, beta-tubulin, microtubule-associated protein-5, microtubule-associated protein-2, and tau after endotoxin stimulation. These results indicate that endotoxin can alter mononuclear phagocyte microtubules by causing an increase in certain microtubule component proteins.
Blotting, Western↗
Monocyte IL-8 release is induced by two independent Fc gamma R- mediated pathways.
Cross-linking of PBMC and monocyte Fc gamma R on immobilized IgG stimulates IL-8 release. We used immobilized anti-Fc gamma R Abs to determine which of the three surface Fc gamma R regulated this IL-8 secretion. Fc gamma RIII cross-linking stimulated PBMC to release 5 times more IL-8 than did either Fc gamma RI or Fc gamma RII clustering (p = 0.001) and stimulated 77% more IL-8 release from PBMC than that from purified monocytes (p = 0.001). In contrast, only Fc gamma RI cross-linking significantly induced monocytes to release IL-8 (p = 0.05). Since purified lymphocytes release little IL-8 in response to immobilized IgG or anti-Fc gamma RIII Abs, we hypothesized that lymphocyte Fc gamma R cross-linking augmented monocyte IL-8 release. Supernatants from IgG- or Fc gamma RIII -stimulated lymphocytes induced monocytes to release more IL-8 than lymphocytes incubated on plastic alone (p = 0.002 and p = 0.003, respectively). THP-1 cells, which do not produce IL-8 in response to Fc gamma i]R cross-linking, also released IL-8 in response to supernatants from IgG- or Fc gamma RIII-stimulated lymphocytes, suggesting that the supernatant activity was not soluble immune complexes. The IL-8-stimulating activity was heat labile, suggesting that the activity is a protein. However, we could not reproduce or block this activity using recombinant cytokines or neutralizing anti-cytokine Abs. Thus, monocyte IL-8 is stimulated directly through Fc gamma RI cross-linking and indirectly through an Fc gamma RIII-stimulated soluble lymphocyte factor.
Cross-Linking Reagents↗
Acute phase levels of C-reactive protein enhance IL-1 beta and IL-1ra production by human blood monocytes but inhibit IL-1 beta and IL-1ra production by alveolar macrophages.
C-reactive protein (CRP), the major acute phase protein in humans, was purified free of endotoxin (LPS) (< 10 pg of LPS/mg of purified CRP) and evaluated for its ability to modulate LPS-induced production of IL-1 beta and IL-1 receptor antagonist (IL-1ra) from human PBMC and lung macrophages. PBMC (5 x 10(6)/ml) released low levels of IL-1 beta in response to either CRP (250 micrograms/ml) or LPS (100 ng/ml) for 18 h (0.3 +/- 0.1 and 1.5 +/- 0.7 ng/ml, respectively). However, when CRP (250 micrograms/ml) and LPS (100 ng/ml) were combined, PBMC released 9.7 +/- 2.9 ng/ml (p < 0.001 vs LPS alone). This synergy was removed by immunodepletion of CRP before stimulation. With respect to IL-1ra, although CRP induced IL-1ra production from PBMC (0.8 +/- 0.3 ng/ml control, 2.6 +/- 1.3 ng/ml with CRP), CRP did not synergize with LPS for IL-1ra production (15.0 +/- 0.7 ng/ml LPS alone vs 15.4 +/- 1.4 ng/ml LPS and CRP). In contrast, lung macrophages responded to CRP quite differently than PBMC. Macrophages (10(6)/ml) were not stimulated to produce IL-1 beta or IL-1ra by CRP alone. When combined with LPS, CRP inhibited IL-1 beta and IL-1ra release induced by LPS (for IL-1 beta release, LPS induced 3.0 +/- 1.7 ng/ml vs 1.1 +/- 0.4 for combined LPS and CRP; for IL-1ra release, LPS induced 12.9 +/- 2.3 ng/ml vs 7.6 +/- 2.3 ng/ml for combined LPS and CRP). These data suggest that acute phase levels of CRP may have divergent effects depending on the target population. CRP may be largely proinflammatory to blood monocytes responding to LPS since IL-1 beta production is augmented over IL-1ra production. However, in tissue compartments the effects of CRP may be largely immunosuppressive to LPS-induced tissue macrophage IL-1 beta production.
Acute-Phase Reaction↗
The combination of endotoxin and dexamethasone induces type II interleukin 1 receptor (IL-1r II) in monocytes: a comparison to interleukin 1 beta (IL-1 beta) and interleukin 1 receptor antagonist (IL-1ra).
Soluble type II interleukin 1 receptor (IL-1r II) and interleukin 1 receptor antagonist (IL-1ra) regulate inflammation by competitively inhibiting the binding of IL-1 beta to the signalling IL-1 receptor. In addition, glucocorticoids also regulate IL-1 beta by suppressing gene transcription. More recently, glucocorticoids have been shown to increase soluble IL-1r II concentrations, which may contribute to their anti-inflammatory properties. Interestingly, increased serum levels of soluble IL-1r II and IL-1ra have been measured in septic patients, although the mechanism is unclear. In this respect, the authors characterize new pathways in which IL-1r II and IL-1ra may be regulated in sepsis through combined stimulation with lipopolysaccharide (LPS) and dexamethasone of peripheral blood mononuclear cells (PBMC). This paper confirms that while dexamethasone induces release of IL-1r II, LPS augments dexamethasone-induced IL-1r II release 45-fold. Furthermore, LPS plus dexamethasone induces IL-1r II protein and mRNA, whereas LPS alone does not. Additionally, it was shown by flow cytometric analysis that the monocyte is the primary IL-1r II producer in response to LPS and dexamethasone administration. Therefore, LPS and dexamethasone synergism in IL-1r II induction may be important in controlling IL-1 beta effects. In contrast, LPS alone induces IL-1ra, while dexamethasone attenuates this LPS-induced response. Although IL-1r II and IL-1ra may work together to suppress IL-1 beta effects in sepsis, inflammatory cells differentially regulate these cytokines.
Blotting, Western↗
Detection of IL-5 and IL-1 receptor antagonist in bronchoalveolar lavage fluid in acute eosinophilic pneumonia.
BACKGROUND: Acute eosinophilic pneumonia is an idiopathic cause of respiratory failure, characterized by very high numbers of alveolar eosinophils without significant blood eosinophilia. OBJECTIVE: The purpose of this study was to determine which cytokines are associated with acute eosinophilic pneumonia. METHODS: Soluble IL-1 type II receptor and the cytokines IL-1 beta, IL-1ra, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha were measured in serum and in bronchoalveolar lavage fluid from two patients with acute eosinophilic pneumonia during both acute and convalescent phases. RESULTS: Compared with patients with adult respiratory distress syndrome, the patients with acute eosinophilic pneumonia had high bronchoalveolar lavage fluid levels of IL-5, IL-1ra, and soluble type II IL-1 receptor but not IL-1 beta, tumor necrosis factor-alpha, IL-3, or granulocyte-macrophage colony-stimulating factor. Bronchoalveolar lavage fluid levels of IL-5 and IL-1ra fell after resolution of symptoms. In the serum of patients with acute eosinophilic pneumonia, IL-5 was not detectable, and IL-1ra was initially high but fell after corticosteroid treatment. CONCLUSION: Acute eosinophilic pneumonia is characterized by locally high levels of IL-5, IL-1ra, and soluble type II IL-1 receptor in the alveolar space.
Acute Disease↗
The pathogenesis of sepsis. Factors that modulate the response to gram-negative bacterial infection.
Gram-negative bacteria gain access to the bloodstream by evading host defenses. Once in circulation, lipopolysaccharide interacts with the host receptor CD14 and initiates the host's immune response. Lipolysaccharide stimulates the host to produce a cascade of mediators that activate and target leukocytes, opsonize the bacteria, and induce fever to defend against the invading bacteria. Unregulated release of these mediators, however, leads to the production of vasoactive substances, activation of the clotting cascade, and diminution of cardiac performance, which leads to the sepsis syndrome. This article discusses the pathogenic events that lead to sepsis syndrome and reviews critical steps in regulating these inflammatory mediators to allow the host to recover from gram-negative bacteremia.
Arachidonic Acids↗
Monocyte Fc gamma receptor cross-linking induces IL-8 production.
In response to bacterial cell wall products such as LPS, monocytes produce IL-8, a powerful neutrophil chemotaxin. However, in the absence of bacterial pathogens, immune complex-mediated diseases such as rheumatoid arthritis are associated with high levels of IL-8 in monocyte-rich compartments. Since it is known that IgG-containing immune complexes can recruit neutrophils via an Fc gamma R-dependent process, we hypothesized that cross-linking of monocyte Fc gamma receptors may induce IL-8. To test this hypothesis, peripheral blood mononuclear cells were evaluated for IL-8 induction in response to immobilized LPS-free pooled human IgG. Immobilized IgG, but not soluble IgG, induced IL-8 in a dose-dependent manner (p < 0.05, r = 0.99). This induction corresponded with an up-regulation in IL-8 steady state mRNA levels that peaked at 4 h. The released IL-8 was functional, since supernatants induced concentration-dependent neutrophil migration that was inhibited by a monoclonal anti-IL-8 Ab. Evaluation of purified monocytes for IL-8 production, as well as FACS analysis of IgG-stimulated PBMC preparations, demonstrated that monocytes are the principal IL-8 producer cell. Thus, monocyte Fc gamma R cross-linking induces biologically active IL-8, which may participate in the pathogenesis of immune complex-mediated diseases.
Cells, Cultured↗
Detection of soluble type II receptor in the presence of its natural ligand IL-1 beta. Quantification by sandwich ELISA.
The type II interleukin-1 receptor (IL-1R II) is a newly described 60-68 kDa protein expressed on monocytes, neutrophils, and lymphocytes. It is hypothesized that a 45 kDa soluble form of the IL-1R II attenuates the proinflammatory effects of IL-1 by preventing its binding to the type I IL-1 receptor. However, very little information exists regarding the detection of soluble IL-1R II. Specifically, there are no reports to date characterizing IL-1R II detection by enzyme-linked immunoassay in the presence of IL-1 beta or characterizing IL-1 beta detection in the presence of IL-1R II. This study addresses the detection and quantitation of IL-1R II and IL-1 beta by a number of sandwich ELISA formats and characterizes the sensitivity of detection in the presence of competitive cytokines. We generated two distinct IL-1R II sandwich ELISAs that can detect receptor down to a level of 50 pg/ml. One, M22/R2, detects only unbound IL-1R II and the other, M2/R2, detects both bound and unbound IL-1R II. In this context, a 4:1 molar ratio of IL-1 beta to IL-1R II interferes with the IL-1R II detection by the M22/R2 but not the M2/R2 ELISA. Conversely, IL-1R II at physiologically relevant concentrations interferes with the detection of IL-1 beta by three distinct IL-1 beta ELISA formats. Taken together, these studies suggest that when measuring samples that may contain both IL-1 beta and IL-1R II, careful attention must be given to assay specificity.
Enzyme-Linked Immunosorbent Assay↗
Taxol and colchicine increase LPS-induced pro-IL-1 beta production, but do not increase IL-1 beta secretion. A role for microtubules in the regulation of IL-1 beta production.
IL-1 beta is a proinflammatory cytokine secreted chiefly by monocytes and macrophages. Currently, much of its mechanism of processing and secretion is poorly understood, but there is increasing evidence that the microtubule system may be involved. For example, it is known that taxol and colchicine, two drugs that affect microtubule structure and function, increase LPS-induced IL-1 beta release. However, it is not known whether these drugs affect the synthesis of the 31-kDa precursor (pro-IL-1 beta) or the processing and release of mature IL-1 beta. To test this, an assay was used that allowed for the temporal separation of IL-1 beta synthesis and release. The addition of taxol or colchicine to the secretory phase of the assay resulted in no significant change in IL-1 beta release. However, when these drugs were added to the stimulus for IL-1 beta production, there was a significant increase in both IL-1 beta release and total IL-1 beta production, as measured by ELISA. These findings indicate that taxol and colchicine increase LPS-induced IL-1 beta release by an increase in the production of the precursor molecule. Thus, it is unlikely that microtubules are involved in the IL-1 beta secretory machinery in any significant manner, but they do play a role in the regulation of pro-IL-1 beta production.
Animals↗
Clinical implications of gene therapy for alpha 1-antitrypsin deficiency.
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Genetic Therapy↗
Immunosuppressive properties of surfactant and plasma on alveolar macrophages.
Alveolar macrophages have been shown to be major producers of the potent proinflammatory cytokines interleukin-1 beta and tumor necrosis factor-alpha, and of the antiinflammatory cytokine interleukin-1 receptor antagonist. During the adult respiratory distress syndrome the normally surfactant-coated alveolus becomes flooded with plasma proteins, altering the milieu of alveolar cells such as alveolar macrophages. To understand alveolar macrophage function during the adult respiratory distress syndrome, the individual and combined effects of surfactant and plasma on alveolar macrophage cytokine production was examined. A synthetic surfactant (Exosurf) and a bovine-derived surfactant (Survanta) both inhibited production of interleukin-1 beta, pro-interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-1 receptor antagonist in a dose-dependent manner. This inhibition was noted when both endotoxin and heat-killed Staphylococcus aureus were used as stimuli. Autologous plasma also inhibited interleukin-1 beta and tumor necrosis factor-alpha release in a dose-dependent manner, but, unlike surfactant, plasma did not inhibit interleukin-1 receptor antagonist release. Similarly, the combination of plasma and surfactant inhibited interleukin-1 beta and tumor necrosis factor-alpha release but not interleukin-1 receptor antagonist release. In support of these data, interleukin-1 receptor antagonist was detectable in five of six bronchoalveolar lavage fluid samples from patients with adult respiratory distress syndrome at a mean concentration of 465 pg/ml; on the other hand, interleukin-1 beta was not detectable in any of these samples. These results indicate that the relative production of interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-1 receptor antagonist can be altered depending on the local concentration of both surfactant and plasma.
Biological Products↗
Fc gamma receptor cross-linking down-regulates IL-1 receptor antagonist and induces IL-1 beta in mononuclear phagocytes stimulated with endotoxin or Staphylococcus aureus.
The cross-linking of monocyte Fc gamma R is a potent stimulus for IL-1ra production but does not induce IL-1 beta. However, during systemic infection, IgG-coated bacteria can activate mononuclear phagocytes via both cell wall components and opsonized IgG. Therefore, we analyzed the effect of combinations of Fc gamma R cross-linking and bacterial cell wall components on mononuclear phagocyte IL-1 beta and IL-1ra production. Human mononuclear cells and monocytes were cultured either alone or with combinations of immobilized IgG, LPS, or heat-killed Staphylococcus aureus (HKSA). Cells cultured on immobilized IgG released large amounts of IL-1ra but no detectable IL-1 beta. In response to LPS, mononuclear cells released IL-1ra at 1000-fold lower doses of LPS than was required to induce IL-1 beta. However, when measured in the presence of immobilized IgG, the LPS sensitivity for IL-1 beta release increased 100-fold, whereas IL-1ra release correlated inversely with the LPS dose. Furthermore, HKSA, a nonendotoxin stimulus, affected mononuclear cell IL-1 beta and IL-1ra release similarly. In addition, polymyxin B, a specific endotoxin inhibitor, blocked the LPS, but not the HKSA-induced changes in IL-1 beta and IL-1ra secretion, irrespective of immobilized IgG co-stimulation. In summary, these results suggest that mononuclear phagocyte stimulation with immobilized IgG favors IL-1ra over IL-1 beta production. Conversely, the addition of LPS or HKSA to the Fc gamma R-stimulated cells augments IL-1 beta but suppresses IL-1ra production.
Cross-Linking Reagents↗
IL-1ra suppresses endotoxin-induced IL-1 beta and TNF-alpha release from mononuclear phagocytes.
The proinflammatory effects of lipopolysaccharide (LPS) are modulated in large part through the induction of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) release by mononuclear phagocytes. However, IL-1's target cell effects can be suppressed by IL-1 receptor agonist (IL-1ra). Because mononuclear phagocytes produce and respond to IL-1 via IL-1 receptors, we hypothesized that IL-1ra may also be able to block receptors on IL-1 producer cells and inhibit secondary IL-1-induced IL-1 production. To test this hypothesis, mononuclear cells and alveolar macrophages were stimulated with LPS in the presence of IL-1ra and analyzed for IL-1 beta and TNF-alpha production. For mononuclear cells, IL-1ra inhibited 6-h LPS- and IL-1 alpha-induced IL-1 beta release to 66 +/- 4% (P = 0.001 by paired t test) and 39 +/- 7% (P = 0.0005 by paired t test) of control, respectively. In addition, IL-1ra reduced both LPS- and IL-1 alpha-stimulated IL-1 beta mRNA levels to 78 and 37% of control, respectively. Furthermore, IL-1ra downregulated both LPS and IL-1 alpha-induced TNF-alpha release to 84 +/- 4% (P = 0.012 by paired t test) and 49 +/- 9% (P = 0.001 by paired t test) of control, respectively. Alveolar macrophages demonstrated variable IL-1ra-induced suppression of LPS-stimulated IL-1 beta and TNF-alpha release.(ABSTRACT TRUNCATED AT 250 WORDS)
Endotoxins↗
Cytokine-induced interleukin-1 receptor antagonist release in mononuclear phagocytes.
IL-1ra has recently been shown to suppress both cytokine- and endotoxin-induced IL-1 beta and TNF-alpha release from monocytes. Given that mononuclear phagocytes can produce both the proinflammatory cytokines IL-1 alpha, IL-1 beta, and TNF-alpha as well as the suppressive cytokine IL-1ra, we proposed that IL-1 alpha, IL-1 beta, and TNF-alpha may induce IL-1ra from mononuclear phagocytes. To test this hypothesis, human mononuclear cells were stimulated for 18 h with IL-1 alpha, IL-1 beta, or TNF-alpha, and the supernatants assayed for IL-1ra by ELISA. Each cytokine induced IL-1ra secretion in a dose-response manner. However, IL-1 alpha and IL-1 beta were better inducers of IL-1ra than was TNF-alpha. IL-1 alpha or IL-1 beta at a dose of 10 ng/ml induced 3 to 6 ng/ml of IL-1ra, while TNF-alpha at a dose of 100 ng/ml stimulated only 1.4 ng/ml of IL-1ra. This induction was not due to endotoxin, as all cytokines contained less than 10 pg/ml of contaminating LPS. Furthermore, for IL-1 beta-induced IL-1ra, immunoprecipitation of IL-1 beta with an anti-IL-1 beta antibody, but not a preimmune antibody, blocked the induction of IL-1ra. In contrast to mononuclear phagocytes, IL-1 alpha, IL-1 beta, and TNF-alpha did not induce further IL-1ra production in alveolar macrophages. This lack of macrophage responsiveness may relate to the constitutive production of IL-1ra by these mature mononuclear phagocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Bronchoalveolar Lavage Fluid↗
Acute occlusion of a mainstem bronchus by a rapidly expanding foreign body.
We report a case of an 85-year-old woman who presented with acute respiratory failure, caused by aspirating a sucralfate tablet that totally occluded her left main-stem bronchus. Acute respiratory failure resolved after bronchoscopic removal of the markedly expanded tablet. To our knowledge, the acute obstruction of a main-stem bronchus by an aspirated foreign body has not been previously described in adults. We believe that the unusual properties of sucralfate tablets (rapid expansion and mucosal binding) were contributing factors in this patient.
Acute Disease↗
Processing proIL-1 beta decreases detection by a proIL-1 beta specific ELISA but increases detection by a conventional ELISA.
Recent investigations have noted that conventional IL-1 beta enzyme linked immunoassays (ELISA) may underestimate proIL-1 beta concentrations. In an attempt to circumvent this problem, we have devised a proIL-1 beta specific ELISA which sandwiches proIL-1 beta between a carboxy-terminus specific, capture antibody, and an amino-terminus specific, detection antibody. The amino-terminus specific antibody was generated against amino acids 3-21 of the intact proIL-1 beta molecule. This sandwich ELISA does not recognize mature 17 kDa IL-1 beta and is not inhibited by the coexistence of mature, 17 kDa IL-1 beta. We compared this proIL-1 beta specific ELISA (amino-terminus ELISA) to the conventional IL-1 beta ELISA (carboxy-terminus ELISA) on test samples that contained either proIL-1 beta or mature IL-1 beta. When purified proIL-1 beta is cleaved by increasing concentrations of IL-1 beta converting enzyme or porcine pancreatic elastase, there is a dose dependent increase in the signal from the conventional IL-1 beta ELISA and a concurrent decrease in signal from the proIL-1 beta specific ELISA. Furthermore, when the proIL-1 beta specific ELISA is used to quantify cellular sources of IL-1 beta, there is no detectable release of proIL-1 beta into the supernatants from either fresh blood monocytes or alveolar macrophages, despite detectable mature IL-1 beta. The cell associated compartment of IL-1 beta is largely proIL-1 beta and the relative amounts of IL-1 beta that remain intracellularly are quite large. Specifically, when assayed at 18 h, the proIL-1 beta ELISA detected 4.6 +/- 1.4 ng/ml per 10(6) monocytes and 13.8 ng/ml per 10(6) macrophages vs. 0.8 +/- 0.3 ng/ml/10(6) monocytes and 1.6 ng/ml/10(6) macrophages by conventional IL-1 beta ELISA. Thus, the 5-10-fold deficit in the detection of intracellular IL-1 beta by a conventional IL-1 beta ELISA, combined with the enhanced detection after enzymatic processing of proIL-1 beta, confirms that a proIL-1 beta specific ELISA is needed to accurately quantify proIL-1 beta.
Enzyme-Linked Immunosorbent Assay↗