The results of a quantitative investigation o f Bcl-2 expression are illustrated in Fig. 6.13 which shows PBMC from an AVI patient stained with Bcl-2-FITC. Various lymphocyte populations were colour coded according to size; red dead cells and debris; green blue and yellow are small, intermediate and large lymphocytes respectively. The background Bcl-2 staining using this reagent was set at 10,000 mol/cell (regions 5,7,9 & 11). From this figure it is clear that as cells increase in size the intensity of Bcl-2 decreases, confirming that the blast-like cells in the peripheral blood of individuals with acute viral infections are low in Bcl-2.
Discussion
Previous studies have shown that the expression of Bcl-2 is closely involved in the regulation of lymphocyte survival associated with selection or deletion of germinal centre B lymphoblasts (Liu et al 1989, Liu et al 1991, Nunez et al 1991) and cortical thymocytes (Hockenberry et al 1991, Sentman et al 1991, Siegel et al
1992). In these tissues the loss of expression o f Bcl-2 is linked with cell death while upregulation of the protein in transgenic mice increases the lifespan o f the cells and can prevent selection (Strasser et al 1991, Siegel et al 1992).
As shown in chapter 4, in both H IV -1^ and AVI patients, activated T cells expressing CD45RO develop in vivo (Froebel et al 1991, Prince & Jensen 1991, Miyawaki et al 1991). These in vivo activated peripheral blood populations were
> ;îm i • I I rnni— i m m in; t—n 1 0 ’ 1 0' 1 0 ' 1 0 bcl2-F!TC m 10 : 10 ® bcl2-FlTC - > r r n r n I I I i | ( I I I | i I I I j 1 1 1 1 1 i n “ i n T | T i T i | i H i I I I I I | i n I j i i i i | i 1 1 1 | l i 10 3C_ 53 -^0 9 0 110
"orw ard Scatter - >
ÏÏDV. a c u t e v i r a l i n f e c t i o n
ID2: 1JAN-1994 b c l - 2 c o n t e t t Total G ated Region P aren t ’/oGate IlnMeanX linStdDevX R1 5000 5000 389 7.78 7.78 M i/ceW R2 5000 5000 973 19.46 19.46 R3 5000 5000 1136 22.72 22.72 R4 5000 5000 810 16.20 16.20 R5 5000 389 132 2.64 33.93 8894.13 2762.19 R6 5000 389 232 4.64 59.64 48633.10 18468.76 R7 5000 973 36 0.72 3.70 10767.35 2901.96 R8 5000 973 915 18.30 94.04 46118.40 16099.80 R9 5000 1136 271 5.42 23.86 9726.41 3225.55 RIO 5000 1136 854 17.08 75.18 39814.34 18558.46 R11 5000 810 303 6.06 37.41 10040.21 2951.07
1
R12 5000 810 459 9.18 56.67 38980.51 18537.921
found to contain a Bcl-2~ component that was expanded compared to normal
individuals. As naive, CD45RA^ T cells develop a primed CD45RO phenotype as a consequence o f stimulation (Sander^ Makgoba & Shaw 1988, Beverley 1990, Akbar et al 1988, Clement 1992) this suggested that Bcl-2 might be down regulated during lymphocyte activation. This possibility was confum ed in isolated C D 45RA ^
lymphocytes which were found to lose Bcl-2 expression during the transition from CD45RA to CD45RO in vitro. Therefore, both acute and chronic in vivo stimulation as well as mitogen induced activation in vitro lead to the development o f a
CD45RO'*' population which lacks Bcl-2.
The C D 8^ lymphocytosis seen in acute viral infection is transient
(Tomkinson et al 1987, Cauda et al 1987, Cheeseman et al 1988, Miyawaki et al 1991) suggesting a rapid clearance of the cells in vivo. Indeed, it has been
demonstrated that these cells die rapidly by apoptosis after culture (Moss et al 1985, Uehara et al 1992). Thus a decrease in Bcl-2 expression renders cells prone to apoptosis and may provide mechanism for the control of T cell numbers after virus induced polyclonal expansion. The correlation between Bcl-2 and apoptosis also holds true for the spontaneous cell death seen in HIV-1 infection. Interestingly, the addition of IL-2 to the activated C D 4 5 R 0 ^ T cells increased Bcl-2 expression and rescued the cells from apoptosis (Akbar et al 1993). Therefore, the lack o f sufficient local levels of IL-2 might lead to a decreased Bcl-2 expression and predispose apoptosis in vivo. Such a scenario might be envisaged in HIV-1 infection where levels of IL-2 are decreased due to C D 4^ T cell depletion.
In contrast, no association between Bcl-2 expression and AALD was found in this study indicating that Bcl-2 is not involved in this form o f cell death.
Additionally, based on morphology and DNA fragmentation studies, AALD could not identified as a classically apoptotic phenomenon. In a recent review on
programmed cell death (PCD) Schwartz & Osborne (1993) noted that PCD and apoptosis are not synonymous. Not all PCD occurs by apoptosis, for instance during insect metamorphosis dying cells do not display the classical signs o f apoptosis, including chromatin condensation and DNA fragmentation. Similarly, apoptosis can occur in the absence o f new gene expression, for instance the DNA fragmentation induced in target cells by cytotoxic lymphocytes (Russell & Dobbs 1980, Duke
Chervenak & Cohen 1983). Therefore, the AALD described here may be another example o f a PCD phenomenon which does not occur by apoptosis /in terestin g ly , one group who noted apoptosis in HIV-1 infected individuals after stimulation with anti-CD3 were unable to show any correlation or increase with disease progression (Meyaard et al 1994). However, levels o f apoptotic cell death were far lower than the AALD described here, further suggesting that AALD is not classical apoptosis. Alternatively, AALD could be an example o f necrotic cell death induced perhaps by some viral protein that interferes with normal cell division. Indeed there is some evidence that dividing cells are affected by HIV-1 infection. For example, many o f the gut and skin complaints o f patients could reflect problems o f epithelial or
epidermal cell regeneration. In addition, patients also suffer hair loss that might also be related to poor cell division. In this respect, viral proteins such as Tat and N ef are known that are known to interfere with normal lymphocyte function (Viscidi et al 1989, Purvis et al 1992) could be implicated.
It still remains a possibility that AALD is also an apoptotic phenomenon but that due to the timing o f the experiment the classical signs were missed. There is only a narrow window during which it is possible to measure DNA fragmentation and if activated cells apoptose at approximately similar times early after stimulation, this might account for the results. Although a daily analysis of cells after stimulation did not reveal extensive apoptosis, a detailed time course involving a more
quantitative assessment of apoptosis, such as the detection o f DNA breaks by in situ
nick translation (Gorczyca et al 1993), might prove useful to validate the findings. Factors other than Bcl-2 protein expression may be involved in cell death after activation. Recently, two bcl-2 related genes box and bcl-X have been reported (Boise et al 1993, Oltval et al 1993). Bcl-X exists in two forms, Bcl-XS and Bcl-XL the latter o f which functions similarly to Bcl-2 and protects cells from apoptosis. The Bax protein forms heterodimers with Bcl-2 and modifies its function (Yin et al
1994). Thus, bcl-X or box may be involved in AALD. In addition, activated C D 4 5 R 0 ^ T cells from individuals with viral infections show increased expression o f Fas/APO-1, indicating that they may be primed for Fas-mediated cell death.
Interleukin-2 is likely to be one of the key molecules involved in keeping activated T cells cycling. However, such continuous stimulation is probably not the
only mechanism whereby mature T cells are maintained. For instance, the co-culture of apoptosis prone lymphocytes with tissue stroma-derived f ib r o b la s tr c ^ extend viability and slow down apoptosis. Studies have shown that the viability o f activated T cells can be extended without significant proliferation after co-culture on
fibroblasts and that they lose their blast-like morphology (Scott, Pandolfi & K um ick 1990). These studies suggest that stromal factors may support the survival o f T cells in a quiescent state and that different microenvironments may influence the fate o f activated T cells.
The greatly expanded C D 8 ^,B cl-2 " population seen in the lymph nodes o f HIV-1 infected individuals may reflect the influence o f stromal factors on T cell survival in vivo as such Bcl-2 negative cells would be expected to die rapidly in the absence of a positive signal. The C D 8 ^,C D 4 5 R 0 ^ lymphocytes in the lymph node show an even more marked loss of Bcl-2 expression than the equivalent population in peripheral blood. Recent data suggests that the lymph node microenvironment is not sufficient to promote the survival o f all of these cells as, using propidium iodide staining, there is clear evidence of apoptosis among the C D 8 ^B cl-2 " population (Bofill et al submitted). Similarly, the huge expansion of C D 8^ cells in the lymph nodes during acute infectious mononucleosis is associated with areas o f necrosis within the tissue. This might be expected if large numbers o f cells are in apoptosis and the macrophages in the paracortex are unable to phagocytose the overwhelming number of dying cells.
This study suggests that the loss of Bcl-2 and subsequent death by apoptosis may be a normal physiological response to pathogen induced proliferation, designed to maintain lymphocyte homeostasis. However, the reasons why cells die after activation are less clear. Studies on cell lines and PBL in vitro point to a role for HIV-1 virus and viral gpl2 0 in cell death by apoptosis (Terai et al 1991, M artin M atear & Vyakamam 1994). The cross-linking o f g p l2 0 on the CD4 molecule, followed by stimulation o f the cell through the TCR causes an activation induced apoptosis of C D 4^ lymphocytes (Banda et al 1992). This may be particularly relevant in vivo in view of the discovery of gpl2 0 in the serum o f HIV-1 patients (Oh et al 1992). Therefore, the loss o f C D 4^ lymphocytes by apoptosis may in part be explained either by infection with HIV-1 or by the presence on the cell surface o f
g p l20 bound to the CD4 molecule. This however does not explain the cell death
observed in the C D 8^ population. '
Interestingly, it was reported by Groux et al (1992) that co-stimulation with anti-CD28 can decrease apoptosis in HIV-1 infection after activation with the superantigen SEE, suggesting that co-stimulation could prevent AALD.