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Pulmonary

Last 50 Pulmonary Postings

(Click on title to be directed to posting, most recent listed first, CME offerings in Bold)

July 2014 Pulmonary Case of the Month: Where Did It Come From?
June 2014 Pulmonary Case of the Month: "Petrified"
May 2014 Pulmonary Case of the Month: Stress Relief
Giant Cell Myocarditis: A Case Report and Review of the Literature
April 2014 Pulmonary Case of the Month: DIP-What?
Wireless Capsule Endo Bronchoscopy
Elevated Tumor Markers In Coccidioidomycosis of the Female Genital Tract
March 2014 Pulmonary Case of the Month: The Cure May Be Worse
   Than the Disease
February 2014 Pulmonary Case of the Month: Faster Is Not Always
   Better
January 2014 Pulmonary Case of the Month: Too Much, Too Late
32 Year Old Man with “Community-Acquired” Pneumonia
December 2013 Pulmonary Case of the Month: Natural
   Progression
November 2013 Pulmonary Case of the Month: Dalmatian Lungs
October 2013 Pulmonary Case of the Month: A Hidden Connection
Bronchoscopic Cryoextraction: A Novel Approach for the Removal
   of Massive Endobronchial Blood Clots Causing Acute Airway
   Obstruction
September 2013 Pulmonary Case of the Month: Chewing the Cud
IgG4-Related Systemic Disease of the Pancreas with Involvement 
   of the Lung: A Case Report and Literature Review
August 2013 Pulmonary Case of the Month: Aids for Diagnosis
Variation in Southwestern Hospital Charges for Pulmonary
   and Critical Care DRGs
July 2013 Pulmonary Case of the Month: Swan Song
June 2013 Pulmonary Case of the Month: Diagnosis
   Makes a Difference
May 2013 Pulmonary Case of the Month: the Cure Can be
   Worse than the Disease
April 2013 Pulmonary Case of the Month: 
   A Suffocating Relationship
Doxycycline Decreases Production of Interleukin-8
   in A549 Human Lung Epithelial Cells
March 2013 Pulmonary Case of the Month:
   Don’t Rein Me In
February 2013 Pulmonary Case of the Month: 
   One Thing Leads to Another
January 2013 Pulmonary Case of the Month: 
   Maybe We Should Call GI
December 2012 Pulmonary Case of the Month: Applying Genetics
November 2012 Pulmonary Case of the Month:
   The Wolves Are at the Door
October 2012 Pulmonary Case of the Month: 
Hemoptysis from an Uncommon Cause
Acetylcholine Stimulation of Human Neutrophil Chemotactic 
   Activity Is Directly Inhibited by Tiotropium Involving Gq Protein
   and ERK-1/2 Regulation
September 2012 Pulmonary Case of the Month:
   The War on Drugs
Tiotropium Bromide Inhibits Human Monocyte Chemotaxis 
August 2012 Pulmonary Case of the Month:
All Eosinophilia Is Not Asthma
COPD Exacerbations: An Evidence-Based Review
July 2012 Pulmonary Case of the Month: Pulmonary Infiltrates -
   Getting to the Heart of the Problem
Cough and Pleural Disease in a Burmese Immigrant – A Masquerader
Meta-Analysis of Self-Management Education for Patients with 
   Chronic Obstructive Pulmonary Disease
June 2012 Pulmonary Case of the Month:
What’s a Millet Seed Look Like?
May 2012 Pulmonary Case of the Month:
   Things Are Not Always as They Seem
April 2012 Pulmonary Case of the Month:
   Could Have Fooled Me!
Pulmonary Nodules with Cutaneous Manifestations: 
   A Case Report and Discussion
March 2012 Pulmonary Case of the Month: There’s Air in There
Treatment of Coccidioidomycosis-associated Eosinophilic
   Pneumonia with Corticosteroids
Sympathetic Empyema Arising from Streptococcus anginosus
   Splenic Abscess
February 2012 Pulmonary Case of the Month
Spirometry Use in Patients Prescribed Albuterol
A 39 Year Old Female with Progressive Dyspnea, 
   Dry Cough and Hypoxia: A Case Report
Congenital Bronchial Atresia: A Case Report With Radiographic 
   And Pathologic Correlation 

 

For complete pulmonary listings click here.

The Southwest Journal of Pulmonary and Critical Care publishes articles broadly related to pulmonary medicine including thoracic surgery, transplantation, airways disease, pediatric pulmonology, anesthesiolgy, pharmacology, nursing  and more. Manuscripts may be either basic or clinical original investigations or review articles. Potential authors of review articles are encouraged to contact the editors before submission, however, unsolicited review articles will be considered.

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Monday
Jul012013

July 2013 Pulmonary Case of the Month: Swan Song

Bridgett Ronan, MD

Lewis J. Wesselius, MD

                                    

Department of Pulmonary Medicine

Mayo Clinic Arizona

Scottsdale, AZ

  

History of Present Illness

A 53 year old man presented to the emergency department with a 2 week history of progressive dyspnea. He thought it was anxiety due to quitting drinking just before the onset of his symptoms. He also had fatigue and malaise.

PMH, SH, FH

He had no significant past medical history or family history. He did not smoke but drank 2-6 beers/day until 2 weeks prior to presentation.

Physical Examination

BP 110/60 mm Hg, P 110 beats/min, R 32 breaths/min, T 37.6° C, SpO2 81%

He is pale and appears mildly dyspneic otherwise his physical exam is unremarkable.

Chest Radiography

His chest x-ray is shown in figure 1.

Figure 1. Initial PA (Panel A) and lateral (Panel B) chest x-ray. 

Which of the following laboratory tests is/are not indicated?

  1. Arterial blood gases
  2. Complete blood count
  3. Spiral thoracic CT angiography
  4. Urinanalysis
  5. All of the above

Reference as: Ronan B, Wesselius LJ. July 2013 pulmonary case of the month: swan song. Southwest J Pulm Crit Care. 2013;7(1):1-9.  doi: http://dx.doi.org/10.13175/swjpcc081-13. PDF

Saturday
Jun012013

June 2013 Pulmonary Case of the Month: Diagnosis Makes a Difference

Lewis J. Wesselius, MD1

Henry D. Tazelaar, MD2

Departments of Pulmonary Medicine1 and Laboratory Medicine and Pathology2

Mayo Clinic Arizona

Scottsdale, AZ

  

History of Present Illness

A 64 year old man from Southern Arizona was referred for a second opinion on a diagnosis of chronic eosinophilic pneumonia that was poorly responsive to corticosteroid therapy. The patient first became ill February 2012 with cough and congestion.  His wife was ill at the same time. Both were treated with antibiotics. His wife improved but he never fully recovered with ongoing symptoms of cough and some dyspnea.

He was admitted to another hospital in August 2012 due to worsening shortness of breath and pulmonary infiltrates on chest x-ray. During this admission he underwent bronchoscopy with bronchoalveolar lavage (BAL) that demonstrated 78% eosinophils. A video-assisted thorascopic (VATs) lung biopsy was done and the patient was diagnosed with chronic eosinophilic pneumonia. He was begun on therapy with high dose prednisone (80 mg/day) but had only slight improvement in symptoms.

He was followed by a pulmonologist and continued on prednisone who questioned the possible development of pulmonary fibrosis. Earlier this year he was started on mycophenolate mofetil and the dose was increased to 1000 mg bid while the prednisone was tapered to 5 mg every other day. He was also being treated with fluticasone/salmeterol 250/50 twice a day. The patient continues to have dyspnea with limited activity. His last pulmonary function testing was done in December 2012. At that time his forced vital capacity (FVC) was 51% of predicted and his diffusing capacity for carbon monoxide (DLco) was 40% of predicted.

PMH, SH, FH

He had a history of obstructive sleep apnea (OSA) and had undergone an uvulopharyngoplasty (UPPP). There was also a history of gastroesophageal reflux disease (GERD) and he had a prior Nissen fundoplication. He had a history of osteoarthritis and had undergone a right shoulder replacement.

He had a remote smoking history, a history of modest alcohol use, but no history of using recreational drugs.  He worked as an airline pilot.

His present medications included mycophenolate mofetil 1000 mg twice a day, prednisone 5 mg every other day, voriconazole 200 mg daily (started after BAL showed a few colonies of Aspergillus), and fluticasone/salmeterol 250/50 twice a day.

Physical Examination

Blood pressure 134/88 mm Hg.  Resting oxygen saturation 96%.

Chest:  bibasilar crackles but no wheezes.

Cardiovascular: the heart had a regular rhythm but no murmur.

Extremities: no clubbing or edema.

The remainder of the physical examination was unremarkable.

Chest Radiography

His chest x-ray is shown in figure 1.

Figure 1. Initial chest x-ray.

Which of the following diseases has/have been associated with increased eosinophils in bronchoalveolar lavage fluid?

  1. Interstitial lung diseases
  2. Acquired immunodeficiency syndrome (AIDS)-associated pneumonia
  3. Idiopathic eosinophilic pneumonia
  4. Drug-induced lung disease
  5. All of the above

Reference as: Wesselius WJ, Tazelaar HD. June 2013 pulmonary case of the month: diagnosis makes a difference. Southwest J Pulm Crit Care. 2013;6(6):247-54. PDF 

Wednesday
May012013

May 2013 Pulmonary Case of the Month: the Cure Can be Worse than the Disease

Lewis J. Wesselius, MD1

Thomas V. Colby, MD2

 

Departments of Pulmonary Medicine1 and Laboratory Medicine and Pathology2

Mayo Clinic Arizona

Scottsdale, AZ

 

History of Present Illness

A 65 year old man from Colorado presented for evaluation of “lung masses.” He had a prior diagnosis of dermatomyositis made in 2010 and had been with intravenous immunoglobulin (IVIG), prednisone and methotrexate. He had been previously seen in January, 2011 with a 5 mm left lower lobe nodule on thoracic CT which was unchanged compared to August, 2010. A thoracic CT scan done in July, 2011 in Colorado was interpreted as stable.

Over the prior month had been having chest discomfort.  He had a history of pulmonary embolism (PE) and felt the pain was similar in quality to his prior PE. This prompted a chest x-ray and he was told of “lung masses”. He had also experienced 20 pound weight loss.

His current medications included methotrexate 25 mg weekly, prednisone 3 mg every other day and warfarin 7 mg daily.

PMH, SH, FH

In addition to dermatomyositis, he has a history of a left lower extremity deep venous thrombosis with PE. At that time protein S deficiency, activated protein C resistance and factor V Leiden mutation were diagnosed and an inferior vena cava filter were placed. He also has a history of paroxysmal atrial fibrillation and the prior lung nodule noted above.

He was a prior smoker, quitting in 1991, but briefly resuming in 2010.  The patient had social alcohol use but no drug use. 

The patient’s father died at age 75 from prostate cancer; his mother died at age 89 with heart disease; and he had a sister living with throat cancer.

Physical Examination

Vital signs: Afebrile; Blood pressure 114/65 mm/Hg; Pulse 80 regular; Oxygen  Saturation  97% on room air at rest

HEENT: limited ability to open mouth

Chest:  few late exp wheezes

CV: Regular rhythm, no murmur

Skin: diffuse erythema, particularly on face. 

Neuro: muscle strength normal 

Radiography

His thoracic CT is shown in Figure 1.

Figure 1. Movies of the thoracic CT scan showing lung windows (Panel A, upper panel) and mediastinal windows (Panel B, lower panel).

Which of the following are pulmonary manifestations of dermatomyositis?

  1. Lung cancer
  2. Aspiration pneumonia
  3. Interstitial lung disease
  4. Metastatic cancer particularly from the cervix, pancreas, breasts, ovaries, gastrointestinal tract and lymph nodes
  5. All of the above

Reference as: Wesselius LJ, Colby TV. May 2013 pulmonary case of the month: the cure can be worse than the disease. Southwest J Pulm Crit Care. 2013;6(5):199-208. PDF

Monday
Apr012013

April 2013 Pulmonary Case of the Month: A Suffocating Relationship

Lewis J. Wesselius, MD

Department of Pulmonary Medicine

Mayo Clinic Arizona

Scottsdale, AZ

  

History of Present Illness

A 70 year old woman from Oregon was referred by urology for evaluation of an abnormal thoracic CT scan. She was asymptomatic.

PMH, SH, FH

She has a prior history of retroperitoneal fibrosis with ureteral obstructions requiring stents and a transient ischemic attack in 2009. During 2012 she developed hypertension and a thoracic CT was done. She has never smoked and is a widowed housewife. There is no family history of lung disease, although her husband died from lung cancer. Her present medications include: amlodipine 10 mg/day, oxybutynin (Ditropan XL) 10 mg/day, and prednisone 5 mg daily.

Physical Examination

Her physical examination was unremarkable.

Radiography

Her chest CT scan is shown in Figure 1.

Figure 1. Thoracic CT movies from mediastinal windows (upper panel) and lung windows (lower panel).

Which of the following is true regarding the CT scan?

  1. There is a right upper lobe mass
  2. There are bilateral pleural effusions
  3. There is lung fibrosis predominately involving the lower lobes
  4. There are diffuse ground glass opacities
  5. There are multiple pleural plaques

Reference as: Wesselius LJ. April 2013 pulmonary case of the month: a suffocating relationship. Southwest J Pulm Crit Care. 2013:6(4):154-60. PDF

Monday
Mar182013

Doxycycline Decreases Production of Interleukin-8 in A549 Human Lung Epithelial Cells*

Jeffrey C. Hoyt1

Janelle G. Ballering1

John M. Hayden1

Richard A. Robbins1,2

 

1Research Service, Carl T. Hayden VA Medical Center Phoenix, AZ,

2Phoenix Pulmonary and Critical Care Research and Education Foundation, Gilbert, AZ

 

Abstract

Doxycycline is an antibiotic that possess anti-inflammatory properties.  These anti-inflammatory properties make doxycycline an attractive candidate for possible treatments for a variety of common chronic obstructive airway diseases. Interleukin-8 (IL-8) is a major inflammatory chemokine and a powerful chemo-attractant for both neutrophils and monocytes.  We hypothesized that doxycycline might exert its anti-inflammatory effects, at least in part, by modulating IL-8 production.  To test this hypothesis, A549 human lung epithelial cells were stimulated with cytomix (IL-1beta, TNF-alpha and gamma-IFN) in the presence or absence of varying concentrations of doxycycline.  Doxycycline decreased IL-8 protein production in a concentration- and time-dependent manner.  In the presence of 30 microg/ml doxycycline IL-8 protein production was decreased by 63% through out a 30 hr time course.  In chemotaxis assays monocyte and neutrophil migration was decreased by 55% and 57% respectively.  Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggest that doxycycline does not decrease expression of IL-8 mRNA and that use of the RNA polymerase II inhibitor DRB indicates that doxycycline does not effect stability of this mRNA.  In the presence of doxycycline p38-alpha mitogen-activated protein kinase (MAPK) expression is decreased by 36% in cytomix-stimulated cells. These data demonstrate that doxycycline can modulate IL-8 release and suggest that it has potential as an anti-inflammatory in those disorders where IL-8 is an important inflammatory mediator.

Introduction

Chemokines are small, excreted, induced proteins that are involved the initiation and regulation of the inflammatory and immunological responses to the presence of foreign antigens in the body.  Interleukin-8 (IL-8) or CXCL8 belongs to the CXC subfamily and is a major pro-inflammatory chemokine produced during chronic inflammatory lung diseases (1-3).  IL-8 performs a variety of functions including activation of degranulation and the respiratory burst in and chemo-attraction of neutrophils (4-6), chemo-attraction and activation of monocytes (7), chemotaxis of T-cells (8), chemotaxis and adhesion of basophils (9,10), activation of 5-lipoxygenase with release of leukotriene B4 (11) and adherence of neutrophils (12) and monocytes (13) to various cell layers.

The common obstructive airway diseases such as bronchitis, asthma, cystic fibrosis, bronchiectasis, chronic obstructive pulmonary disease (COPD) and diffuse panbronchiolitis are all invariably associated with chronic airway inflammation (14-16).  These disorders are often progressive even with current therapies, including steroid therapy, suggesting that new, more effective therapies are needed.

The tetracyclines and erythromycin are well known, potent antibacterial agents.  In addition both compounds also possess a separate anti-inflammatory mode of action and have been known to reduce inflammation associated with several inflammatory diseases since the 1970’s (17,18).  These compounds include erythromycin A, and many of its derivatives such as azithromycin and clarithromycin, as well as tetracycline and several related compounds such as doxycycline.  These compounds have exhibited anti-inflammatory activity in a variety of inflammatory disorders (18).

We hypothesized that doxycycline possesses potent anti-inflammatory properties, and that treatment with doxycycline leads to a reduction in IL-8 levels in A549 human lung epithelial cells. Such a reduction would lead to an attenuation of many pro-inflammatory responses and possibly limiting tissue damage at the site of inflammation.  Our results show that doxycycline decreases the production of IL-8 in cytomix (a combination of IL-1 beta, TNF-alpha and gamma-IFN) stimulated cells resulting in decreased monocyte and neutrophil chemotaxis.  These results suggest that doxycycline may be beneficial in the control of the inflammatory response in a variety of chronic airway disorders.

Materials and Methods

Culture of A549 Cells. The human lung epithelial cell line, A549, was purchased from the American Type Culture Collection (Manassas, VA) (19). A549 cells were grown in 25 cm2 tissue culture flasks (Corning Costar, Cambridge, MA) in Ham's F‑12 containing 10% fetal calf serum, L‑glutamine (2 mM) and penicillin‑streptomycin (100 U/ml‑100 mg/ml) until confluent.  After washing with serum-free media doxycycline hydrochloride, dissolved in distilled water, was added to confluent A549 cultures for 3 hr and the cells were cultured 18 hr in serum-free Ham's F-12 with or without cytomix.  Cytomix (CM) is a combination of recombinant human tumor necrosis factor-alpha (TNF-alpha), human interleukin‑1 beta (IL-1 beta), and human interferon-gamma (IFN-gamma) each at a 5 ng/ml concentration (all cytokines from R&D Systems, Minneapolis, MN) that have been previously used to stimulate the expression of IL-8 (20,21).  Concentrations of doxycycline ranged from 3 to 30 microg/ml and are based on reports of clinically relevant serum and tissue levels (22).  All control cultures contained the appropriate amount of carrier solvent alone.

Cell Viability Assays.  Cell viability was determined by assay for release of lactic acid dehydrogenase into cell culture supernatant fluid using a commercially available assay kit (Sigma, St. Louis, MO) according to the manufacturer’s directions.

Determination of IL-8 Protein Levels.  IL-8 protein levels were determined using a commercially available ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s directions.  Doxycycline concentrations, cytomix stimulation and culture supernatant collection times were performed as indicated in the figure legends.

Neutrophil and Monocyte Chemotaxis. To perform the neutrophil chemotaxis assay, polymorphonuclear leukocytes were purified from heparinized normal human blood by the method of Böyum (23). The resulting cell pellet routinely consists of > 96% neutrophils and > 98% viable cells as determined by trypan blue and erythrosin exclusion. The cells were suspended in Hanks' balanced salt solution (HBSS) containing 2% bovine serum albumin (BSA, Sigma) at pH 7.4 to give a final concentration of 3.0 x 106 cells/ml. This suspension was used for the neutrophil chemotaxis assay.

Mononuclear cells for the chemotaxis assay were obtained by Ficoll-Hypaque density centrifugation to separate red blood cells and neutrophils from mononuclear cells. This preparation routinely consisted of 30% large monocytes and 70% small lymphocytes determined by morphology and alpha-naphthyl acetate esterase staining with >98% viability as assessed by trypan blue and erythrosin exclusion. The cells were resuspended in HBSS containing 2% bovine serum albumin at pH 7.4 to give a final cell concentration of 5 x 106 cells/ml.

The chemotaxis assay was performed in a 48-well micro-chemotaxis chamber (NeuroProbe Inc., Cabin John, MD). Briefly, 25 μl of the harvested cell culture supernatant fluids were placed into the lower wells and a 10-µm thick polyvinylpyrrolidone-free polycarbonate filter  (Nucleopore, Pleasanton, CA), with a pore size of 3 μm  for neutrophil chemotaxis or 5 µm for monocyte chemotaxis, was placed over the bottom wells. The silicon gasket and upper pieces of the chamber were applied and 50 μl of the appropriate cell suspension added to the upper wells above the filter. The chambers were incubated in humidified air in 5% CO2 at 37°C for 30 min for neutrophil chemotaxis or 90 min for monocyte chemotaxis. After incubation, the chamber was disassembled and non-migrated cells were wiped away from the filter. The filter was then immersed in methanol for 5 min, stained with Diff-Quik (American Scientific Product, McGraw Park, IL), and mounted on a glass slide. Cells that completely migrated through the filter were counted by using light microscopy in ten random high power fields (HPF) per well.

RNA Isolation and RT-PCR.  IL-8 mRNA was analyzed by reverse transcription‑polymerase chain reaction (RT‑PCR). Total cellular RNA was extracted from adherent cells using a modification of the methods of Chomczynski and Sacchi (24).  The RNA was reverse transcribed using a commercially available RT-PCR kit (Promega, Madison, WI) and commercially available IL-8 primers (R&D Systems, Minneapolis, MN) added at 0.2 μM final concentration. Using a Perkin Elmer model 480-thermal cycler, reverse transcription was performed on 250 ng of total cellular RNA at 48oC for 45 min.  PCR for IL-8 was performed in the same sample tubes at 94°C for 2 minutes followed by 22 cycles consisting of 94°C for 45 sec, 55°C for 45 sec, 72°C for 2 min, followed by 72°C for an additional 7 min.  Beta-actin was used as a “housekeeping gene" and RT-PCR was performed in a similar manner as for IL-8 except for an annealing temperature of 55°C. The beta-actin primer sequences that were used are:  sense primer 5’-TGACCCAGATCATGTTTGAG-3’ and antisense primer 5’-TCATGAGGTAGTCAGTCAGG-3’.  The DNA fragments were separated by agarose gel electrophoresis on a 2% gel in TAE buffer and then identified, analyzed and quantified by densitometry.  Increasing PCR cycles gave increasing amounts of DNA through a fairly broad range with linearity obtained from about cycles 22-28 beginning with 250 ng of DNA (r=0.99 for IL-8 and beta-actin).

Stability of IL-8 mRNA.  A549 cells were treated with cytomix or cytomix plus doxycycline for 18 hr as described above and then treated with the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizaole (DRB) (Calbiochem, La Jolla, CA), 10 μg/ml, to inhibit transcription.  RNA was isolated from cultures at various times and RT-PCR, using conditions described previously, was used to determine the amount of IL-8 mRNA present at indicated times.

Protein Determination.  Protein concentrations were determined spectrophotometrically using the Bradford assay (25).

Western Blotting. Cell extracts were prepared for Western blotting using previously described methods (26).  Cell lysates were clarified by centrifugation prior to electrophoresis.  SDS-PAGE was performed using a 4% T acrylamide stacking gel and a 10% T separating gel.  Twelve micrograms of protein were loaded per lane.  After electrophoresis was complete the separated proteins were transferred to a PVDF membrane for further analysis. Transfer was performed using a Tris-HCl (25 mM), glycine (192 mM) and methanol (20% v/v) buffer system.  The transfer was performed at 4oC for 16 hr at 30V and then for 30 min at 60V.  Antibody for p38-alpha MAPK (Santa Cruz Biotechnology, Santa Cruz, CA) was used to immunochemically detect p38-alpha MAPK protein on the transfer membranes using goat anti-rabbit IgG alkaline phosphatase as the secondary antibody-enzyme conjugate and NBT/BCIP alkaline phosphatase substrate tablets (Sigma, St. Louis, MO) in 0.1 M Tris-HCl pH 9.1 as the substrate system.   Five microg of antibody were used at each step during the development procedures.  The transfer membranes were blocked with BLOTTO (20 mM Tris base, 180 mM NaCl, 4% nonfat dry milk, 0.02% NaN3) to minimize nonspecific binding.

 

Statistical analysis. In cases where multiple experiments were made, differences between groups were tested for significance using one-way analysis of variance (ANOVA) with Fisher’s protected least significant difference (Fisher’s PLSD).  In cases where single measurement was made, the differences between groups were tested for significance using Student’s paired t-test.  In all cases, a p value of <0.05 was considered significant. The data are expressed as mean + SD.

Results

A549 cell culture supernatants from all treatments were assayed for lactic acid dehydrogenase release as a measure of cell viability.  There were no significant differences in the lactic acid dehydrogenase activity in the culture supernatants from all treatment types (data not shown).

Cytomix stimulation of A549 cells results in an increase in IL-8 production from 1100 pg/ml in unstimulated control samples to > 230,000 pg/ml (Fig. 1). 

Figure 1.  Effect of doxycycline concentration on production of IL-8.  Cytomix (CM) with or without 3-30 microg/ml doxycycline (Dox). A549 cells were treated, samples collected and IL-8 ELISA assays were performed as described in Materials and Methods section.  Results are from at least three experiments with each condition repeated in triplicate.  * p< 0.01.

Doxycycline alone at all concentrations tested did not significantly increase IL-8 production over control samples.  Increase in doxycycline concentration in cytomix-stimulated cells results in a concentration-dependent decrease in IL-8 production.  At the highest doxycycline concentration used (30 microg/ml) IL-8 production was decreased 60% over cytomix treatment alone (p < 0.01) (Fig. 1). 

In a time course study 30 microg/ml doxycycline treatment of cytomix-stimulated cells suppressed IL-8 production starting at 2 hr and continuing to the end of the study at 30 hr (p < 0.01 at all times).  Suppression ranged from 70% at 2 hr to 80% at 30 hr (p < 0.01) (Fig. 2).

Figure 2.  Time course for IL-8 production in the presence of cytomix (CM) and 30 microg/ml doxycycline (Dox).  A549 cells were treated and IL-8 ELISA assays were performed as described in Materials and Methods section. Results are from at least three experiments with each condition repeated in triplicate.  Hatched columns are CM alone.  Black columns are CM + Dox.  *In all cases p < 0.01.

Doxycycline treatment decreased neutrophil chemotaxis by 57% in cytomix-stimulated cells relative to cytomix alone (p < 0.01) (Fig 3A).  In companion monocyte chemotaxis experiments doxycycline decreased chemotaxis by 55% relative to cytomix alone (p < 0.01) (Fig. 3B).

Figure 3.  Effect of doxycycline on: (A) neutrophil chemotaxis and (B) monocyte chemotaxis.  Results show the number of cells that migrate into the chemotaxis membrane in each microscope field of view examined. At least five high power microscope fields were counted for each treatment. * In all cases p < 0.01.

Expression of IL-8 mRNA was not affected by the presence of 30 microg/ml doxycycline.  At this doxycycline concentration, cytomix-induced IL-8 mRNA expression is not altered relative to samples treated with cytomix alone (Fig. 4).  Doxycycline alone did not increase IL-8 mRNA expression over untreated control samples (Fig. 4).

Figure 4.  Effect of doxycycline on IL-8 and beta-actin mRNA production.  Total cellular RNA was isolated and RT-PCR was performed as described.  IL-8 RTPCR Panel A:  Lane 1 base pair markers.  Lane 2: 30 μg/ml doxycycline alone.  Lane 3: 4 hr cytomix (CM).  Lane 4: 4 hr CM + 30 μg/ml doxycycline.  Lane 5:  12 hr CM.  Lane 6: 12 hr CM + 30 μg/ml doxycycline. Lane 7:  18 hr CM.  Lane 8: 18 hr CM + 30 μg/ml doxycycline.  Lane 9: 30 hr CM.  Lane 10: 30 hr CM + 30 μg/ml doxycycline.  Lane 11: base pair markers.  Lane 12: IL-8 positive control.  Beta-actin RTPCR panel B: Lane 1: base pair markers.  Lane 2: 30 μg/ml doxycycline alone.  Lane 3: 4 hr cytomix (CM).  Lane 4: 4 hr CM + 30 μg/ml doxycycline.  Lane 5: 12 hr CM.  Lane 6: 12 hr CM + 30 μg/ml doxycycline. Lane 7:  18 hr CM.  Lane 8: 18 hr CM + 30 μg/ml doxycycline.  Lane 9: 30 hr CM.  Lane 10: 30 hr CM + 30 μg/ml doxycycline.  Results are from duplicate experiments with each condition assayed in triplicate.

Stability of IL-8 mRNA in cytomix-stimulated cells does not appear to be affected by doxycycline (Fig. 5).  IL-8 mRNA appears to decrease at the same rate, as indicated by the nearly identical slopes of the decay lines, in the presence or absence of doxycycline over an 8 hr time course.

Figure 5.  .  IL-8 mRNA stability.  Total cellular RNA was isolated in the presence of 10 microg/ml DRB at the indicated times and RT-PCR performed as described.  Solid line:  cytomix alone (r =0.967).  Dashed line:  CM + 30 microg/ml doxycycline (r = 0.645).  Results are from triplicate experiments.          

The effect of doxycycline p38-alpha MAPK was examined (Fig. 6).  Treatment of cytomix-stimulated cells with 30 microg/ml doxycycline decreased p38-alpha protein production by 36% over cytomix-stimulation alone.

Figure 6.  Effect of doxycycline on p38 alpha MAPK.  Cells were lysed and electrophoresis and blotting were performed as described.  Lane: (1) Molecular weight markers (kDa), (2) Control,  (3) Doxycycline (30 microg/ml) alone, (4) CM  alone,  (5) CM  plus doxycycline (30 microg/ml).  20 microg of protein loaded per lane.

Discussion

IL-8 is a major component of the many protein-based chemoattractant molecules that occur in chronic, inflammatory airway diseases (27).  As a chemoattractant it recruits both monocytes and neutrophils to inflamed areas of the body.  We have shown that doxycycline-treatment of cytomix-stimulated A549 human lung epithelial cells results in a concentration-dependent decrease in IL-8 production (Fig. 1) as well as in a time-dependent decrease in IL-8 production (Fig. 2).  The observed decrease in IL-8 production affects neutrophil (Fig. 3A) and monocyte (Fig. 3B) chemotaxis.  Chemotaxis of both neutrophils and monocytes are decreased after doxycycline treatment of stimulated cells indicating a possible reduction in the inflammatory response.  Expression (Fig. 4) and stability (Fig 5) of IL-8 mRNA appear to be unaffected in cytomix-stimulated cells after doxycycline treatment.  The p38 MAPK protein production is also decreased in the presence of doxycycline (Fig. 6).

The lack of effect of doxycycline of IL-8 mRNA expression and stability suggests that the observed decrease in IL-8 protein production may be due to post-translational events and not directly related to regulation of gene activity through involvement of activator protein-1 (AP-1), nuclear factor-kappa beta or other transcriptional regulatory systems.  These events could possibly be a variety of post-translational modifications, such as glycosylation of specific amino acid residues in the IL-8 protein, incorrect or lack of removal of the N-terminal hydrophobic signal peptide, or by increased proteolytic degradation of IL-8 by the proteasome, matrix metalloproteases or other proteolytic activity.  Another intriguing possibility is the report by Li et al. (28) that decreased p38 MAPK activity has no effect of IL-8 gene transcription but that active p38 MAPK plays a critical role in IL-8 protein production in a post-transcriptional manner.  This observation appears to describe the decreased IL-8 and p38 MAPK protein levels and the apparent lack of effect on IL-8 mRNA that we have described.

IL-8 is an important piece of the inflammatory response to invasion of the body by various foreign objects.  However, if the inflammatory response is allowed to progress unchecked substantial cell and tissue damage occurs. 

IL-8 production occurs in a wide variety of inflammatory lung diseases that are characterized by an influx of neutrophils (1).  Asthma is often thought of as a disease that is dependent on T-lymphocytes and eosinophils (1,29).  However there is mounting evidence that neutrophils and IL-8, a powerful chemoattractant for neutrophils, are elevated in severe asthma cases (29).  Diffuse panbronchiolitis (DPB) is a progressive, chronic, inflammatory airway disease that is largely restricted to Japan (30).  Neutrophils, T-lymphocytes and the chemoattractant molecules IL-8 and MCP-1 are thought to play significant roles in the progression of DPB (30).  Cystic fibrosis is characterized by a massive influx of neutrophils into the airways (31).  In addition neutrophil chemoattractants such as IL-8 are found in very high concentration (31).  In COPD IL-8 is a prominent attractant for neutrophils (32-Barnes) and IL-8 levels in induced sputum correlate with disease severity (32).  A common result for these chronic, inflammatory lung diseases is severe, often life threatening, tissue destruction. 

Continuous treatment with some antibiotics, particularly macrolides, reduces exacerbations. A randomized controlled trial with erythromycin reduced exacerbations by 35% compared to placebo (33). In a more recent study, treatment with azithromycin for one year lowered exacerbations by 27% (34). Although the mechanism(s) accounting for the reduction in exacerbations is unknown, current concepts suggest the reduction is likely secondary to the macrolides’ anti-inflammatory properties. However, concern has been raised about a very small, but significant, increase in QT prolongation and cardiovascular deaths with azithromycin (35). In addition, the recent trial with azithromycin raised the concern of hearing loss which occurred in 25% of patients treated with azithromycin compared to 20% of control (34).

Tetracycline and derivatives such as minocycline and doxycycline have been extensively used to treat a wide variety of chronic, inflammatory diseases (36) including acne, rosacea, rheumatoid arthritis and periodontitis.  Nieman and Zerler (37) propose that tetracycline and some of its derivatives may be beneficial in limiting lung damage during adult respiratory syndrome (ARDS).  Although macrolide antibiotics such as erythromycin A have been shown to be effective treatments for cystic fibrosis (38) and diffuse panbronchiolitis (39) development of resistant pathogenic bacteria during the required prolonged therapy is a major concern.  Doxycycline is not the antibiotic of choice for many respiratory bacterial infections so development of resistance to this drug is of less importance than it is for the macrolides.

Systemic inflammation has been correlated with poorer outcomes in COPD (40). Doxycycline has anti-inflammatory properties in addition to suppressing release of IL-8 including reduction of or inducible nitric oxide production and MCP-1 release from lung epithelial cells (41,42). It seems likely that doxycycline might reduce other inflammatory cytokines as well. These results suggest that doxycycline may provide a unique, effective, low cost therapy for management of a wide range of chronic, inflammatory diseases.

References

  1. Pease JE, Sabroe I.  The role of interleukin-8 and its receptors in inflammatory lung disease:  Implications for therapy.  Am J Respir Med. 2002; 1:19-25.
  2. Hay DW, Sarau HM.  Interleukin-8 receptor antagonists in pulmonary diseases.  Curr Opin Pharmacol. 2001; 3:242-247.
  3. de Boer WI.  Cytokines and Therapy in COPD.  Chest. 2002; 121:209S-218S.
  4. Mukaida N.  Interleukin-8: an expanding universe beyond neutrophil chemotaxis and activation.  Int J Hematol. 2000; 72:391-398.
  5. Sato E, Simpson KL, Grisham MB, Koyama S, Robbins RA.  Reactive nitrogen and oxygen species attenuate interleukin-8-induced neutrophil chemotactic activity in vitro.  J Biol Chem. 2000; 275:10826-10830.
  6. Willems J, Joniau M, Cinque S, van Damme J. Human granulocyte chemotactic peptide (IL-8) as a specific neutrophil degranulator: comparison with other monokines.  Immunology. 1989; 67:540-542.
  7. Traves SL, Smith SJ, Barnes PJ, Donnelly LE.  Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2.  J Leukocyte Biol. 2004; 76:441-450.
  8. Larsen CG, Anderson AO, Appella E, Oppenheim JJ, Matsushima K.  The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes.  Science. 1989; 243:1464-1466.
  9. Geiser T, Dewald B, Ehrengruber MU, Clark-Lewis I, Baggiolini M.  The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes.  J Biol Chem. 1993; 268; 268:15419-15424.
  10. Bacon KB, Flores-Romo L, Aubry JP, Wells TN, Power CA.  Interleukin-8 and RANTES induce the adhesion of the human basophilic cell line KU-812 to human endothelial cell monolayers.  Immunology. 1994; 82:473-481.
  11. Schroder JM.  The monocyte-derived neutrophil activating peptide (NAP/interleukin-8) stimulates human neutrophil arachidonate-5-lipoxygenase, but not the release of cellular arachidonate.  J Exp Med. 1989; 170:847-863.
  12. Carveth HJ, Bohnsack JF, McIntyre TM, Baggiolini M, Prescott SM, Zimmerman GA.  Neutrophil activating factor (NAF) induces polymorphonuclear leukocyte adherence to endothelial cells and to subendothelial matrix proteins.  Biochem Biophys Res Commun 1989; 162:387-393.
  13. Srinivasan S, Yeh M, Danziger EC, Hatley, ME, Riggan AE, Leitinger N, Berliner JA, Hedrick CC. Glucose regulates monocyte adhesion through endothelial production of interleukin-8.  Circ Res. 2003; 92:371-377.
  14. National Heart Lung and Blood Institute/World Health Organization. 2002.  NIH Publication No. 02-3659.
  15. National Heart Lung and Blood Institute/World Health Organization. 2013.  http://www.goldcopd.org
  16. Kharitonov SA, Barnes PJ.  Exhaled markers of inflammation. Curr Opin Allergy Clin Immunol. 2001; 1:217-224.
  17. Avila PC, Boushey HA.  Macrolides, asthma inflammation and infection. Ann Allergy Asthma Immunol. 2000; 84:565-568.
  18. Labro MT. Immunomodulation by antibacterial agents.  Is it clinically relevant?  Drugs. 1993; 45:319-328.
  19. Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP.  In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors.  J Natl Cancer Inst. 1973; 51:1417-1423.
  20. Robbins RA, Springall DR, Warren JB, Kwon OJ, Buttery LD, Wilson JJ, Adcock IM, Riveros-Moreno V, Moncada S, Polak J, Barnes PJ.  Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation.  Biochem Biophys Res Commun. 1994; 198:835-843.
  21. Jones KL, Bryan TW Jinkins PA, Simpson KL, Grisham MB, Owens MW, Milligan SA, Markewitz BA, Robbins RA.  Superoxide causes a reduction in nitric oxide gas and an increase in nitrate. Am J Physiol. 1998; L1120-L1126.
  22. Hartnett BJ, Marlin GE.  Doxycycline in serum and bronchial secretions.  Thorax. 1976; 31:144-148.
  23. Böyum A.  Isolation of mononuclear cells and granulocytes from human blood.  Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g.  Scand J Clin Lab Invest. 1968;97(Suppl):77-89.
  24. Chomczynski P, Sacchi N.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987; 162:156-159.
  25. Bradford MM.  A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding.  Anal Biochem. 1976; 72:248-254.
  26. Robinson VK, Sato E, Nelson DK, Camhi SL, Robbins RA, Hoyt JC.  Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro.  Free Radic Biol Med. 2001; 30:986-991.
  27. Sato E, Koyama S, Takamizawa A, Masubuchi T, Kubo K, Robbins RA, Nagai S and Izumi T.  Smoke extract stimulates lung fibroblasts t release neutrophil and monocyte chemotactic activities.  Am J Physiol. 1999; 277:L1149-L1157.
  28. Li J, Kartha S, Iasvovskaia S, Tan A, Bhat RK, Manaligod JM, Page K, Brasier AR and Hershenson MB.  Regulation of human airway epithelial cell IL-8 expression by MAP kinases.  Am J Physiol Lung Cell Mol Physiol. 2002; 283:L690-L699.
  29. Gibson PG, Simpson JL and Saltos N.  Heterogeneity of airway inflammation in persistent asthma:  evidence of neutrophilic inflammation and increased sputum interleukin-8.  Chest. 2001; 119:1329-1336.
  30. Poletti V, Chilosi M, Casoni G and Colby TV.  Diffuse panbronchiolitis.  Sarcoidosis Vasculitis and Diffuse Lung Diseases 2004; 21:94-104.
  31. Berger M.  Inflammatory mediators in cystic fibrosis lung disease.  Allergy and Asthma Proc. 2002; 23:19-25.
  32. Barnes PJ.  Mechanisms in COPD: differences from asthma.  Chest. 2000; 117:10S-14S.
  33. Seemungal TA, Wilkinson TM, Hurst JR, Perera WR, Sapsford RJ, Wedzicha JA. Long-term erythromycin therapy is associated with decreased chronic obstructive pulmonary disease exacerbations. Am J Respir Crit Care Med. 2008;178:1139-47.
  34. Albert RK, Connett J, Bailey WC, Casaburi R, Cooper JA Jr, Criner GJ, Curtis JL, Dransfield MT, Han MK, Lazarus SC, Make B, Marchetti N, Martinez FJ, Madinger NE, McEvoy C, Niewoehner DE, Porsasz J, Price CS, Reilly J, Scanlon PD, Sciurba FC, Scharf SM, Washko GR, Woodruff PG, Anthonisen NR; COPD Clinical Research Network. COPD Clinical Research Network. Azithromycin for prevention of exacerbations of COPD. N Engl J Med. 2011; 365:689-98.
  35. Ray WA, Murray KT, Hall K, Arbogast PG, Stein CM. Azithromycin and the risk of cardiovascular death. N Engl J Med. 2012;366:1881-90.
  36. Sapadin AN and Fleischmajer R.  Tetracyclines: nonantibiotic properties and their clinical applications.  J Am Acad Dermatol. 2006; 54:258-265.
  37. Nieman GF and Zerler BR.  A role for the anti-inflammatory properties of tetracyclines in the prevention of acute lung injury.  Curr Med Chem. 2001; 8:317-325.
  38. Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibenen DA, Coquillette S, Fieberg AY, Accurso FJ and Campbell PW, III.  Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa.  JAMA. 2003; 290:1749-1756.
  39. Tamaoki J, Kadota J and Takizawa H. Clinical implications of the immunomodulatory effects of macrolides.  Am J Med. 2004; 117 Suppl 9A:5S-11S.
  40. Agustí A, Edwards LD, Rennard SI, MacNee W, Tal-Singer R, Miller BE, Vestbo J, Lomas DA, Calverley PM, Wouters E, Crim C, Yates JC, Silverman EK, Coxson HO, Bakke P, Mayer RJ, Celli B; Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) Investigators. Persistent systemic inflammation is associated with poor clinical outcomes in COPD: a novel phenotype. PLoS One. 2012;7(5):e37483.
  41. Hoyt JC, Ballering J, Numanami H, Hayden JM and Robbins RA.  Doxycycline modulates nitric oxide production in murine lung epithelial cells.  J Immunol. 2005; 176:567-572.
  42. Raza M, Ballering JG, Hayden JM, Robbins RA and Hoyt JC. Doxycycline decreases monocyte chemoattractant protein-1 in human lung epithelial cells.  Exp. Lung Res. 2006; 32:15-26.

*Funded by the Flight Attendants Medical Research Institute (FAMRI)

 Reference as: Hoyt JC, Ballering JG, Hayden JM, Robbins RA. Doxycycline decreases production of interleukin-8 in a549 human lung epithelial cells. Southwest J Pulm Crit Care. 2013;6(3):130-42. PDF

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