Apoptotic cell death, detected ex vivo in peripheral blood lymphocytes of HIV-1 infected persons

In HIV-1 infection the ongoing depletion of CD4+ T-lymphocytes is believed, to a large extent, to be due to apoptosis. Until now quantitative information about in vivo apoptosis of lymphocytes in HIV-patients is scarce because of the very nature of the apoptotic process. Successful detection of apoptosis ex vivo requires the recognition of the initial phase of this process, because at a later stage the cells may not remain any longer in the circulation. We measured quantitatively the amount of early apoptotic peripheral blood lymphocytes directly ex vivo in HIV-1 infected patients using a recently described flow cytometric assay. With this method we observed in an unselected heterogenous group of twelve HIV-infected individuals a median percentage of apoptotic lymphocytes to be significantly higher than in ten healthy controls. To the best of our knowledge this is the first report of ex vivo observed increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.

IN HIV-1 infection the ongoing depletion of CD4+ T-lymphocytes is believed, to a large extent, to be due to apoptosis. Until now quantitative information about in vivo apoptosis of lymphocytes in HIV-patients is scarce because of the very nature of the apoptotic process. Successful detection of apoptosis ex vivo requires the recognition of the initial phase of this process, because at a later stage the cells may not remain any longer in the circulation. We measured quantitatively the amount of early apoptotic peripheral blood lymphocytes directly ex vivo in HIV-1 infected patients using a recently described flow cytometric assay. With this method we observed in an unselected heterogenous group of twelve HIVinfected individuals a median percentage of apoptotic lymphocytes to be significantly higher than in ten healthy controls. To the best of our knowledge this is the first report of ex vivo observed increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.

HIV infection
Apoptotic cell death, detected ex vivo in peripheral blood lymphocytes of HIV-1 infected persons Introduction HIV-1 infection leads to a gradual depletion of CD4+ T-lymphocytes. This phenomenon cannot be explained solely by a direct cytopathic effect of HIV since the frequency of HIV infection is as minor as 1 in 100000 cells in early infection. 1'2 Evidence is accumulating that the CD4+ cell loss is due to apoptosis to a large extent and that apoptosis of peripheral blood lymphocytes is a general feature in HIV-infected persons. This important phenomenon is not limited to the CD4+ population because in advanced HIV-1 infection both CD4+ and CD8+ cells are primed for the cell death programme, by overexpression of the Fas antigen, a P4hysiological receptor for apoptosis signalling. Calculations of the turnover rate of peripheral CD4+ lymphocytes in HIV-1 infected patients resulted in estimates of 3 10 7 cells per day suggesting a high apoptotic rate. 5 These data could not been substantiated by direct measurements because, until now, assays that provide quantitative information about apoptosis lack specificity or sensitivity, are time consuming and usually require the destruction of cell integrity.

Subjects and Methods
We measured quantitatively apoptosis of peripheral blood lymphocytes ex vivo in twelve HIV-1 infected individuals and in ten healthy controls using the APOPTESTTM-FITC protocol (NeXins Research, the Netherlands), which numerates early apoptotic cells by probing for cell surface exposed phosphatidylserine (PS) with FITC labelled annexin and for plasma membrane integrity by propidium iodide (PI) exclusion. This test discriminates between intact cells (FITC-/PI-), early apoptotic (FITC+/ PI-) and necrotic cells (FITC+/PI+) as described elsewhere. 6'7 Procedures followed were in accordance with the policies of the Institutional Ethical Review Board of the Hospital Group.

Results
The results of twelve consecutively measured peripheral blood samples of patients are given in Table 1. The amount of early apoptotic cells is expressed as percentage and absolute count of FITC-positive and PI-negative lymphocytes being flow cytometric recognized by their In HIV-infected patients this paradoxically induces apoptosis instead of proliferation as the result of an inappropriate reemergence of the programmed cell death pathway as described after in vitro stimulation. 12 Our experience shows that the annexin V assay is a feasible and relatively simple method for ex vivo detection of early apoptotic lymphocytes.
To the best of our knowledge this is the first report of ex vivo observed increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.