1. INTRODUCTION

 The study of lymphocyte susceptibility to programmed cellular death (PCD) is acquiring more and more clinical importance. There is indeed a growing evidence that an increased susceptibility to PCD plays a pivotal role in several pathologies like autoimmune diseases and viral infections.

Usually, the first step to perform cytometric evaluation of apoptosis is the separation of peripheral blood mononuclear cells (PBMC) through a density gradient. This procedure allows the removal of granulocytes and red blood cells, but can generate several drawbacks, among which we can remember: i) consistent waste of time, ii) necessity of manipulating dangerous biological samples, iii) selective loss of cell subpopulations, and iv) cell activation by mechanical stimulation.

With the aim to overcome these problems, we set up a technique able to evaluate PBMC spontaneous susceptibility to PCD in short term whole blood cultures. The proponed method consists of a modification of Nicoletti's technique, and is based on multiparametric FACS analysis of physical parameters, DNA content and CD15 expression of peripheral blood leucocytes cultured for 24 or 48 h in RPMI 1640 medium supplemented with fetal calf serum (FCS).

A threshold set on forward scatter together with an electronic window selective for events with CD15 negativity allows to focus the analysis on a cellular subset mainly made up of PBMC (Figs. 1-3).

After properly setting the analysis gate, it is possible to carry out DNA analysis in this selected cell population, defining as apoptotic the cells with a hypodiploid DNA content.

Multiparametric analysis shows that the forward scatter of cells with hypodiploid DNA content is clearly different from cells with diploid content (fig. 2). This behavior is well-known, and is related to the cell shrinkage induced by the apoptotic process

Finally, the result of the analyses can be expressed as percentage of apoptotic cells, or as apoptotic index, i. e. the ratio between vital and apoptotic cells.

 

         2.1 Materials

a) RPMI 1640, supplemented with 20% Fetal Calf Serum (FCS) (A2);
b) Fetal Calf Serum (A2);
c) Phosphate Buffered Saline (PBS) (A2);
d) NH₄Cl 0,84% (A1, A2);
e) Ethanol 70% (A1, A2);
f) Propidium iodide (PI) 10 µg/ml (A1, A2);
g) Monoclonal Antibody anti CD15, FITC conjugated, clone MMA (A2);
h) Flow cytometer (A3);
i) CO₂ water jacketed incubator (A3).
 

2.2 Methodology

 

1) Blood samples were collected in sterile heparynized tubes, and cultures were performed delivering 1 ml of whole blood in 9 ml of 20% FCS-supplemented RPMI 1640. Cultures were cultured in a 95% humidified atmosphere with 5% CO₂ at 37°C.

2) At 24 and 48 h respectively, 1 ml of cell suspension was drawn from the culture, washed with cold PBS, resuspended in 100 µl of PBS containing 20 µl of anti-CD15 FITC-conjugated MoAb, and incubated for 20 min at 4°C.

3) Two ml of hypotonic NH₄Cl solution were then added, and cells incubated for 10 min at room temperature, washed in cold PBS and resuspended in 2 ml of cold 70% ethanol for at least 1 h.

4) Finally, cells were washed in cold PBS, and resuspended in 1 ml isotonic fluorochrome solution (PI 10mg/ml in PBS). Cells were analyzed after a minimum of 30 min of incubation in this solution. Flow cytometry was performed using a FACScan flow cytometer.

 

 

3.1 Background information

Briefly, this method consists of a first level, low cost and easy to perform technique able to explore the susceptibility to PCD of whole blood PBMC.

Instead of removing granulocytes and red blood cells by centrifugation through density gradient, this technique focuses the DNA analysis on a multiparametric defined cell population. Its rationale is based on a widely accepted point, namely that the extrusion of apoptotic bodies gives the apoptotic cell a hypodiploid content of DNA. Its main clinical application could be the exploration of susceptibiliy to apoptosis in PBMC from patients with autoimmune or viral diseases; nonetheless, an application in the evaluation of peripheral blast response to anticancer drugs is possible.

This technique can be easily improved in a dual laser system using a red-excitable dye (e.g. ToPro3) for DNA analysis, and introducing more blu-excitable phenotypic or metabolic probes for further characterization of subsets interested by the apoptotic process. Such modifications are currently in development in our Laboratory.

 
3.2 Critical parameters

No critical parameters were found with the described procedure.

 
3.3 Troubleshooting.

Lower FCS concentration can result in an increase of debris, making difficult the isolation of lymphocyte cluster on physical parameters.

 
3.4 Anticipated results

In a group of 40 healthy persons (blood donors), the mean apoptotic index was 0.04 (range 0.01-0.09) at 24 h, and 0,06 (range 0.03-0.15) at 48 h. A significative linear correlation (r=0.62) was found between values measured at 24 and at 48 h (data not shown).

 

3.5 Time considerations

This method is very simple and fast. The time needed for the setting up of cultures is negligible, and the time requested by sample staining and analysis is similar to an ordinary immunophenotypic FACS analysis. Moreover, when resuspended in ethanol, the sample can be stored for several days at +4°C without perceptible loss of fluorescence.

 

3.6 Key references.

 

1) Carbonari, M., M. Cibati, M. Cherchi, D. Sbarigia, A. M. Pesce, L. Dell'Anna, A. Modica, and M. Fiorilli. 1994. Detection and characterization of apoptotic peripheral blood lymphocytes in Human Immunodeficiency Virus infection and cancer chemotherapy by a novel flow immunocytometric method. Blood. 83:1268-1277.

2) Cossarizza, A., C. Mussini, N. Mongiardo, V. Borghi, G. Kalachnikova, B. De Rienzo, and C. Franceschi. 1997. Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome. AIDS. 11:19-26.

3) Emlen, W., J. A. Niebur, and R. Kadera. 1994 Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J. Immunol. 152:3685:3692.

4) Endresen, P. C., P. S. Prytz, and J. Aarbakke. 1995. A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA. Cytometry. 20:162-171.

5) Gougeon, M.-L., H. Lecoeur, A. Dulioust, M.-G. Enouf, M. Crouvoisier, C. Goujard, T. Debord, and L. Montagnier. 1996. Programmed cell death in peripheral lymphocytes from HIV-infected persons. Increased susceptibility to apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with disease progression. J. Immunol. 156:3509-3520.

6) Jaleco, A. C., M. J. Covas, and R. M. M. Victorino. 1994. Analysis of lymphocyte cell death and apoptosis in HIV-2-infected patients. Clin. Exp. Immunol. 98:185-189.

7) Lorenz, H.-M., M. Grünke, T. Hieronymus, M. Herrman, A. Kühnel, B. Manger, and J. R. Kalden. 1997. In vitro apoptosis and expression of apoptosis-related molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases. Arthritis Rheum. 40:306-317.

8) McCloskey, T. W., N. Oyaizu, M. Coronesi, and S. Pahwa. 1994. Use of a flow cytometric assay to quantitate apoptosis in human lymphocytes. Clin. Immunol. Immunopathol. 71:14-18.

9) Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, and C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods. 139:271-279.

10) Ormerod, M. G., F. Paul, M. Cheetham, and X. M. Sun. 1995 Discrimination of apoptotic thymocytes by forward light scatter. Cytometry. 21:300-304.

11) Pandolfi, F., M. Pierdominici, A. Oliva, G. D'Offizi, I. Mezzaroma, B. Mollicone, A. Giovannetti, L. Rainaldi, I. Quinti, and F. Aiuti. 1995. Apoptosis-related mortality in vitro of mononuclear cells from patients with Hiv infection correlates with disease severity and progression. J. Acquir. Immune. Defic. Syndr. 9:450-458.

 

 

Solution Preparation Storage
 

a) NH₄Cl 0,84% in H₂O Room Temperature
b) Ethanol 70% in H₂O +4°C tightly capped
c) Propidium Iodide 10 µg/ml in PBS Room Temperature in darkness
 

Caution! PI is toxic, mutagen and carcinogen.

 

 Appendix 2: Reagents

 

a) Ethanol, cod 983, Merck (Darmstadt, Germany);
b) Fetal Calf Serum, cod S0113-1, Seromed Biochrom KG (Germany);
c) Monoclonal Antibody anti CD15, FITC conjugated, cod 347423, Becton Dickinson (San Josè, CA, USA);
d) NH₄Cl, cod A4514, Sigma (St. Louis, MO, USA);
e) Phosphate Buffered Saline, cod 1000-3, Sigma (St. Louis, MO, USA);
f) Propidium Iodide, cod P4170, Sigma (St. Louis, MO, USA).
g) RPMI 1640 Tissue Culture Medium, cod 5087-72-8, Difco Laboratories (Detroit, MI, USA).

 

 

Appendix 3: Equipment

 

a) Five parameter flow cytometer, argon laser operated (FACScan Becton Dickinson, Mountain View, CA, U.S.A.);

b) CO2 incubator, Heraeus Instruments GmbH (Hanau, Germany).

 

Appendix 4: Glossary
 
 
SCATTER: Collective response of a particle to an incident beam, resulting from interaction between reflection and refraction phenomena. Light scatter collected at a narrow angle from the incident beam (forward scatter, or FSC) can be considered proportional to the cell size, whereas light scatter collected at 90° (side scatter, or SSC) can be considered proportional to the cell granularity.

 
Figure 1-3. Cytograms produced by a typical analysis in a subject with HIV-1 infection.


Fig. 1, left panel: analysis of the physical parameters; R1, R2 and R3 identify the regions of lymphocytes/monocytes, granulocytes and apoptotic cells, respectively.
Fig. 1, right panel: the staining with FITC-labelled anti-CD15 mAb allows a clear identification of granulocytes (those present in R2 in the previous panel).
 
 
 


Fig. 2, left panel: simultaneous analysis of DNA content and CD15 expression in all cells;
Fig. 2, right panel: analysis of DNA content and SSC in CD15- cells (i.e. those present in R1 and R3).
 
 



Fig. 3: histogram referred to CD15- cells which shows a classical hypodiploic peak.