Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 3, 1995, pp. 163-164

REPORTE CORTO / SHORT REPORT

THE IFN ganma PATHOPHYSIOLOGY. THE ROLE OF SOLUBLE IFNganmaR alpha CHAIN

Yraldo Bello^1, A. Martinto^1, Marisela Suarez^1, Giselle Penton^1, Isabel Apezteguia^1, O. Fernandez^1, Alfredo Menendez^2; Michel Aguet^3 and Pedro Lopez Saura^1.

^ 1Department of Cellular Biology. Center for Biological Research, La Habana 6, P.O.Box 6996. ^2Genetic Engineering and Biotechnology, La Habana 6, P.O. Box 6162. C.P. 10600, Cuba. ^3Molecular Biology I. Univ. Zurich, Honggerberg, Zurich 8093, Switzerland.

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Some experimental findings point to a disease-promoting role of IFNganma in several pathological status as multiple sclerosis, systemic lupus erithematosus, type I diabetes, septic shock and others. In the case of rheumatoid arthritis (RA) specially the route of administration are of critical importance in determining the effects of IFNganma. We showed for the first time the altered levels of the IFNganmaR alpha chain in patients with AR and some RFLP of genomic DNA. The IFNganmaR alfa chain soluble fragment was cloned and expressed in E. coli.

INTRODUCTION

IFNganma is a lymphokine produced by T cells and NK cells. IFNganma exerts its activities by binding to specific cell surface receptor (R).

IFNganma acts as a potent immunomodulator and is a powerful macrophage activating factor. In the basis of its properties the IFNganma could play the principal role in the antigen specific immune response and has been considered the master key to the inflammatory response (1).

Some experimental findings point to a disease-promoting role of IFNganma in several pathological status as multiple sclerosis, systemic lupus erithematosus, type I diabetes, septic shock and others (2). In the case of rheumatoid arthritis (RA) specially the route of administration are of critical importance in determining the effects of IFNganma.

It is possible that at sites of inflammation the pro-inflammatory properties of IFNganma predominate, whereas critical concentration of circulating IFNganma are anti-inflammatory.

An aberrant regulation of the IFNganma action at site of inflammation could contribute to the development and/or exacerbation of some autoimmune and inflammatory disorders.The control of IFNganma function at the receptor level could be an important place to elucidate the possible disregulation of IFNganma system in IFNganma promoting diseases.

We showed for the first time the altered levels of the IFNganmaR alpha chain in patients with AR and some RFLP of genomic DNA. The IFNganmaR alfa chain soluble fragment was cloned and expressed in E. coli.

EXPERIMENTAL PROCEDURES

Soluble IFNganmaR alpha chain was isolated from plasma of RA patients and controls using minichromatographies on IFNganma coupled to affigel-10 columns. Messenger RNA and genomic DNA were isolated from PBL after CsCl gradient, and processed for Northern Blot and RFLP analysis respectively.

The recombinant IFNganmaR alpha chain soluble fragment was isolated by cDNA-PCR using specific primers. The cDNA was inserted in a pIL-2 expression vector. The recombinant protein was extracted with Guanidium chloride 7M and then refolded by gel filtration. The protein was purified sequentially by anion exchange chromatography and ligand affinity.

RESULTS

Soluble forms of IFNganmaR alpha chain were identified in human plasma by dot blot with ^125I-IFNganma. The soluble receptor migrated as 68kDa in reduced conditions and as 60 kDa in non-reduced conditions.

The levels of soluble receptor in RA patients and controls detected by dot blot is showed in table.

A possible RFLP was identified affecting the 5' coding region for IFNganma binding site in the IFNganmaR alpha chain. The recombinant IFNganmaR alpha chain soluble fragment was expressed at high level in E. coli. The maximum of expression was at 8 h after induction by starvation of tryptophan. The recombinant protein recognized ^125I-IFNganma in a dot blot and Western Blot and non-labeled IFNganma in an ELISA system.

CONCLUSIONS

An altered soluble receptor molecule with high affinity or high levels of the soluble molecule could generate an aberrant regulation of IFNganma function during the primary events that contribute to the development of RA. The use of recombinant IFNganmaR alpha chain soluble fragment is a good candidate as antagonist to IFNganma at site of inflammation and will be helpful in counteract the proinflammatory action of the IFNganma.

REFERENCES

1. BILLIAU, A. and R. DUKMANS, (1990) Interferon gamma: mechanism of action and therapeutic potential. Bioch. Pharmacol. 40: 1433-1439.

2. KURSCHNER, C.; G. GAROTTA and Z. DEMBIC (1992) Construction, purification and characterization of new interferon (IFN) inhibitor proteins. J. Biolg. Chem. 267: 9354-9360.

Table 1

% of relative high levels of IFNgammaRalpha chain

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Populations       Control     Suspected      Patients
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Swiss           14.3% (1/7)   60% (3/5)    35% (5/14)
Cuban           11.7% (2/17)   -           20% (1/5)
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