Neutrophil Chemotactic Factors in Bacterial Pneumonia

doi:10.1378/chest.95.5.1021

Chest

Volume 95, Issue 5, May 1989, Pages 1021-1027

https://doi.org/10.1378/chest.95.5.1021 Get rights and content

The influx of neutrophils into the lung is a prominent feature in patients with bacterial pneumonia. Since neutrophils migrate in response to chemotactic factors, chemotactic activity was evaluated in bronchoalveolar lavage (BAL) fluid obtained from 12 patients with bacterial pneumonia and ten normal control subjects. Chemotactic activity was greatly elevated in the BAL fluid of the pneumonia patients compared with control subjects (p<0.01). To partially characterize the chemotactic factors present in the lavage fluid of the patient group, molecular sieve chromatography was performed on the lavage fluid, and at least three peaks of chemotactic activity were identified. Since the molecular weight of the smaller peaks approximated the molecular weight of two known chemotactic factors, C5a and leukotriene B4, these factors were measured in lavage fluid by radioimmunoassay. C5a was detectable in none of the normal subjects but was detectable in four of 14 BAL samples obtained from the patients. Leukotriene B4 was detectable in all subjects and was significantly elevated in the pneumonia patients (552 ± 95 vs 81 ± 16 pg/ml, p<0.01). These findings demonstrate that elevated neutrophil chemotactic activity is present in the lungs of patients with bacterial pneumonia and suggest that C5a and leukotriene B4 may account, at least in part, for this increase.

Section snippets

Patient Population

Bacterial pneumonia was diagnosed in 12 patients by the following criteria: (1) clinical pneumonia with fever, dyspnea, and productive cough; (2) chest roentgenograms with areas of consolidation; and (3) isolation of a potential bacterial pathogen from sputum and/or BAL fluid. For comparison, ten normal subjects were also evaluated. Each normal subject had normal results of physician examination, chest roentgenogram, and pulmonary function testing.

Bronchoalveolar Lavage

To sample the lower respiratory tract,

Patient Characterization and BAL Findings

The characteristics of the patients with pneumonia are presented in Table 1. The patients were significantly older than the normal subjects (66 ± 17 vs 30 ± 6 years, p<0.01). Further, all the patients with pneumonia had underlying conditions that complicated their pneumonia, including six with COPD and one each with head trauma, emphysema, diabetic ketoacidosis, Parkinson’s disease, congestive heart failure, fracture of the hip, and a seizure disorder.

Bronchoalveolar lavage was performed

DISCUSSION

An influx of neutrophils into the alveoli is a characteristic pathologic finding in bacterial pneumonia.1, 2, 3 Since current concepts suggest that neutrophils migrate to chemotactic factors and that neutrophils are critical to the host in destroying and clearing these pathogenic bacteria,4, 5, 6, 7, 8, 9, 10, 11, 12 the mechanisms which cause the migration of neutrophils from the vasculature into the involved alveoli are important in understanding the host defenses against bacterial pneumonia.

ACKNOWLEDGMENTS

The writers thank Ms. Linda Goodman and Ms. Lillian Richards for secretarial assistance and Dr. Martha A. Mellencamp for reviewing the manuscript.

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We have developed and validated label-free, liquid chromatography–mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.
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However, additional pulmonary diseases are possibly associated with LTs (Peters-Golden and Henderson, 2007; Taccone-Gallucci et al., 2008). LTB4 levels have been shown to be increased also in broncho-alveolar lavage fluid from human patients with bacterial pneumonia (Hopkins et al., 1989), as well as in rat and murine models of pneumonia (Buret et al., 1993; Bailie et al., 1996). The results of the present study show that a 5-LOX-dependent biochemical pathway is activated also in different acute and chronic porcine pneumonia, which are caused by specific microorganisms, being characterized by typical gross and microscopic lesions, and likely resulting from different pathogenetic mechanisms.
Enzootic pneumonia by Mycoplasma hyopneumoniae and pleuropneumonia by Actinobacillus pleuropneumoniae are among the most common and economically relevant pulmonary diseases in swine herds. We herein investigated the activity and expression of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) in healthy and diseased porcine lungs, by means of immunohistochemical, immunochemical and biochemical assays. Diseased lungs showed a significantly higher activity and expression of 5-LOX and COX-2 in a wide range of cell types, thus suggesting the likely involvement of both enzymes in the pathogenesis of bacterial porcine pneumonia. Consistently, increased enzyme activities were paralleled by increased leukotriene B₄ (LTB₄), a 5-LOX product and prostaglandin E₂ (PGE₂), a COX-2 product, content in diseased versus healthy lungs.
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Moreover, it has been shown that these mediators are involved in antimicrobial host defense in a variety of infectious diseases [7–9]. Elevated levels of LTB4 are found in the bronchoalveolar lavage fluid of patients with bacterial pneumonia [10] and the levels of LTB4 and leukotriene C4 (LTC4) are similarly elevated in lung homogenates of animals with experimental bacterial pneumonia [7]. These findings provide evidence of 5-LO pathway activation during the course of lower respiratory tract infections.
Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B₄ and the anti-inflammatory substance lipoxin A₄ by ∼50%, increased 60-day mortality from 14% to ∼57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by ∼15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) – presumably leukotrienes – positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB₄) rather than a suppressor (LXA₄) of antimicrobial defense, suggesting the major effect of leukotrienes.
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Methods: Retrospective chart review. The microbiologial diagnosis was obtained by BAL culture or/and urinary antigen assay.
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Two receptors for leukotriene B₄ (LTB₄) have been molecularly identified: BLT1 and BLT2. Both receptors are G protein-coupled seven transmembrane domain receptors, whose genes are located in very close proximity to each other in the human and mouse genomes. The two receptors differ in their affinity and specificity for LTB₄: BLT1 is a high-affinity receptor specific for LTB₄, whereas BLT2 is a low-affinity receptor that also binds other eicosanoids. The two receptors also differ in their pattern of expression with BLT1 being expressed primarily in leukocytes, whereas BLT2 is expressed more ubiquitously. By mediating the activities of LTB₄, these receptors participate both in host immune responses and in the pathogenesis of inflammatory diseases. Reduced disease severity in animal inflammatory models seen with LTB₄ receptor antagonists and in mice with targeted deletion of BLT1 have revealed important roles for LTB₄ and its receptors in regulating pathologic inflammation.

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Supported in part by a grant from the Veterans Administration.

Manuscript received May 20; revision accepted September 9.

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Chest

Neutrophil Chemotactic Factors in Bacterial Pneumonia

Section snippets

Patient Population

Bronchoalveolar Lavage

Patient Characterization and BAL Findings

DISCUSSION

ACKNOWLEDGMENTS

Pathogenesis and pathology of lobar pneumonia

Lancet

Studies on the mechanism of recovery in pneumococcal pneumonia: I. The action of type specific antibody upon the pulmonary lesions of experimental pneumonia

J Exp Med

Studies on the cellular immunology of acute bacterial infections

Harvey Lect

Granulocyte transfusion therapy of experimental Pseudomonas pneumonia

J Clin Invest

Infection during induction of remission in acute lymphocytic leukemia

Cancer

Quantificative relationships between circulating leukocytes and infection in patients with acute leukemia

Ann Intern Med

Infections in patients with cancer

Semin Oncol

Causes of death in adults with acute leukemia

Medicine (Baltimore)

The relationship of fever, granulocytopenia and antimicrobial therapy to bacteremia in cancer patients

Cancer

Therapy of infections in patients with granulocytopenia

Med Clin North Am

Fever and infection in leukemic patients: a study of 494 consecutive patients

Cancer

Three antibiotic regimens in the treatment of infection in febrile granulocytopenic patients with cancer

J Infect Dis

Use of sequential bronchoalveolar lavage to enrich for “bronchial” and “alveolar” material [Abstract]

Clin Res

Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage

J Lab Clin Med

Inflammatory and immune processes in the human lung in health and disease

Am J Pathol

A 48-well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration

J Immunol Methods

Isolation of mononuclear cells and granulocytes from human blood

Scand J Clin Invest

Biologically active peptides of complement: techniques and significance of C3a and C5a measurement

A radioimmunoassay for leukotriene B4

Prostaglandins

Estimation of volume of epithelial lining fluid recovered by lavage using urea as a marker of dilution

J Appl Physiol

Activation of the complement system in the adult respiratory distress syndrome

Am Rev Respir Dis

Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation

Science

Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophore A23187

Proc Natl Acad Sci USA

Serum coated zymosan stimulates the synthesis of leukotriene B4 in human polymorphonuclear leucocytes: inhibition by cyclic AMP

Biochem Biophys Res Commun