MIP-Iβ (CCL4): Estudio de su regulación transcripcional y evaluación de las implicaciones del polimorfismo a nivel de locus B

Abstract

The chemokines are a small group of structurally related molecules that regulate cell trafficking of various types of leukocytes in physiologic and pathologic conditions. In general the chemokines can be divided into four groups based on a structural cysteine motiff found near to the amino-terminus of the mature protein. The four groups are as follows: The CXC or a-chemokines, the CC or b-chemokines, the C or g-chemokine and finally the CX3C or d-chemokine. Into the b-chemokines groups, we found MIP-1a and MIP-1b chemokines, that are clustered at chromosome 17 q 11.2.
The chemokines exert their biological functions trough binding to seven transmembrane-domain receptors wich are expressed on a variety of leukocyte populations. Three receptors has been described for MIP-1b: CCR5, CCR8 and CCR9, being CCR5 who give it antiviral properties, because it is one of the VIH-1 coreceptors.
Human MIP-1b in encoded by two loci, locus A and locus B, and the last one define a polymorphism in this chemokine. This polymorfic form is characterized by a deletion of the first 15 bp in exon 3 and its caused by a mutation A260-G, origining a protein that lacks 5 amino acids. In order to determine the healthy population incidence of this polymorphism, we have studied their allelic distribution in 200 donors using the PCR-SSP technique, confirmed by PCR-SSO technique post-hibridization and sequentiation, showing that the frequency of the canonic form represent a 83,5% whereas a polymorphic form represent a 16,5%. On the other hand, this study was done in a population of thyroid autoimmune disseasse patients, diabetes patients and , hepatitis C patients showing that exist a correlation between genomic frequency and allelic frequency that these patienes with the healthy population. However, the VIH patients showed differences statistically significant respect to the healthy population.
In the study of the level expression of MIP-1a and MIP-1b, we used a protocol that, taking advantage of small difference in nucleotide sequence, combines RT-PCR and restriction with MspI to allow the semiquantitative assessment of mRNAs from the two chemokines. The key feature of our approach is the introduction of an internal standard common to MIP-1a and MIP-1b wich is co-amplified and contains a MspI restriction site. This allow and easy control of the amplification and the efficiency of the restriction. This method was used to determine the possible difference in the level expression of the loci A and B of MIP-1b in the cellular line U-937 after its stimulation with PMA, LPS and Ionomicine, as well as between healthy populations and VIH patients (of different genotype) in stimulated PBMCs and lymphocytes TCD8 with PHA and IL-2.
The study show that the loci A and B are regulated of different way in the cellular line U-937, and the mRNA cinetics expression are equivalents in VIH patients and healthy populations PBMCs and lymphocytes TCD8. In general terms, the locus B contribute in a minoritary way to the synthesis of the mRNA of MIP-1b and the induction cinetic of the mRNA of the lymphocytes TCD8 are deleted in heterozigous and homozigous (bb).
On the other hand, the expression levels of CCR5 receptor in lymphocytes TCD4 are different to the controls.
Finally, we can conclude that MIP-1b acts in a different way in a target cells due to a different iso- and alo-forms that it present.

Date of Award	11 Oct 2002
Original language	Spanish
Supervisor	Manuel Juan Otero (Director)