Procoagulant And Inflammatory Response of Virus-Infected Monocyes

Provided by:

Enrique Gurfinkel , MD, PhDFundación Favaloro, Capital Federal, Buenos Aires, Argentina

Editorial Slides VP Watch – November 13, 2002 - Volume 2, Issue 45

Introduction

• Atherosclerosis is thought to be an inflammatory disease, but its origin still remains unanswered.

• Chronic low-grade inflammation and combination of classical risk factors, plus novel risk factors such as the infectious burden seem to contribute to promote atherosclerosis.

Introduction

• Monocytes play a prominent role in inflammation, coagulation and the ability to produce tissue factor and cytokines.

• All of these factors, particularly the production of cytokines may contribute to acute coronary syndromes by eliciting plaque instability.

Background

• During viral infections monocytes predominantly produce inflammatory cytokines.

• In addition, continuing viral infection may sustain excess tissue factor production and exhaust the inhibitory effects of tissue factor pathway inhibitor.

As highlighted in VP Watch of this week,

• Bouwman and co-workers tried to establish whether virus-infected monocytes initiate coagulation.

And

• To detect if the production of cytokines by monocytes may contribute to the chronic process of atherosclerosis in an in-vitro model.

Methods

• Isolation of monocytes: monocytes obtained from fresh blood of healthy volunteers.

Methods• Preparation of virus stocks: Human embryonic

lung, human epidermoid larynx carcinoma, and LLC-MK2 cells were cultured in Eagles´s minimum essential medium with Earle´s salts.

• A clinical isolete of CMV was propagated in HEL cells

• Chlamydia pneumoniae strain AR 39 was propagated in HEP-2 cells

• Influenza A HIN1 86 Singapore was propagated in LLC-MK2 cells

Methods

• Coagulation assay: Normal pooled plasma was prepared from the blood of nine healthy donors.

• Cytokine assays: IL 6, 8, and 10 were measured in the supernatants of virus-infected monocytes.

It appeared that all strains could infect monocytes, but in all cases the infection was below 5% when undiluted virus stocks were used.

Fluorescence microscopy images of virus-infected monocytes. Magnification 200x after overnight incubation; Red background: uninfected monocytes. Green areas: virus-infected monocytes stained with FITC-labeled anti-virus antibodies. (a) CMV, specific staining of CMV in the nuclei of the infected cells. (b) Influenza A, smooth staining pattern of influenza A in monocytes. (c) Cp, more dense staining pattern of Cp in the cytoplasm of monocytes.

80% of the monocytes were covered with activated CD41+ CD42+

Ficoll + VPT+ VPT+Depletion Depletion Positive Selection

CD41+ CD42+ 46% 78% 3%

CMV and Cp, like influenza, reduced the clotting time.The inititation of coagulation by virus-infected monocyes

was a result of the expression of TF.

Clo

ttin g

tim

e (s

econ

d s)

Uninfected monocytes Infected monocytes

140+-22.1 s

333+-22.1s

Infection with CMV and Cp induced the production ofmodest levels of IL6 and IL8, whereas infection with influenza A

strongly stimulated the production of IL6 and 8.IL

-6 I L

-8 ( p

g m

l)

CMV & Cp

IL 6

1000

IL-8

H1N1

2000

Discussion

• The results of the present study indicate that only small quantities of an infectious virus are needed to stimulate monocytes to exert considerable immunological effects.

Conclussions

• The procoagulant activity of virus-infected monocytes is TF-dependent.

• Influenza infection induced a pronounced expression of IL-6 and IL-8, which could be associated with plaque rupture.

Questions

• Is there any differences between the bacterial and viral infectious burden in promoting procoagulant activity?

• Is it plausible to think that this could explain a systemic pro-thrombotic process after infection?

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6. Frostegard J, Ulfgren A, Nyberg P, Hedin U, Swedenborg J et al. Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage stimulating cytokines. Atherosclerosis 1999;145:33–43.

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8. Bouwman J. J. M. et al,. Eur J Clin Invest 2002; 32 (10): 759-766

References