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Activation of adherent vascular neutrophils in the lung during acute endotoxemia

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Abstract

Background

Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature.

Methods

Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity.

Results

Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein.

Conclusion

These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells.

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Abbreviations

BAL:

bronchoalveolar lavage

BSA:

bovine serum albumin

C/EBP:

CCAAT/enhancer binding protein

COX-2:

cyclooxygenase-2

CREB:

cAMP response element binding protein

FBS:

fetal bovine serum

fMLP:

n-formyl-methionyl-leucyl-phenylalanine

HBSS:

Hank's balanced salt solution

ICAM-1:

intercellular adhesion molecule-1

LPS:

lipoplysaccharide

MAP:

mitogen activated protein

MIP-2:

macrophage inflammatory protein-2

NF-κB:

nuclear factor-kappa B

PBS:

phosphate-buffered saline

PI3K:

phosphatidyl inositol 3 kinase

PKB:

protein kinase B

RT-PCR:

Reverse transcription-polymerase chain reaction

TBE:

Tris borate EDTA

TNF-α:

tumor necrosis factor alpha

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Acknowledgements

This work was supported by NIH Grants TGES04738, ES06897, ES05022 and GM34310 and U.S. Public Health Service Grant EY09056 and NIEHSTG ES07148.

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Rutgers University, 160 Frelinghuysen Road, 08854-8020, New Jersey, Piscataway, USA

    Vasanthi R Sunil, Agnieszka J Connor, Peihong Zhou, Marion K Gordon & Debra L Laskin

  2. Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA

    Jeffrey D Laskin

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  1. Vasanthi R Sunil
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  2. Agnieszka J Connor
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  3. Peihong Zhou
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Correspondence to Debra L Laskin.

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Sunil, V.R., Connor, A.J., Zhou, P. et al. Activation of adherent vascular neutrophils in the lung during acute endotoxemia. Respir Res 3, 14 (2002). https://doi.org/10.1186/rr171

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