61

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_4, © Springer Science+Business Media New York 2012

    Chapter 4   

 Assessment of Mitochondrial Dysfunction in Lymphocytes 
of Patients with Systemic Lupus Erythematosus       

         Andras    Perl        ,     Robert    Hanczko   ,  and     Edward    Doherty       

  Abstract 

 Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within 
the immune system. Activation, proliferation, or death of cells of the immune system is dependent on 
controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mito-
chondrial transmembrane potential (

Δ

  y   

m

 ) re fl ects the energy stored in the electrochemical gradient across 

the inner mitochondrial membrane which, in turn, is used by F 

0

 F 

1

 -ATPase to convert ADP to ATP during 

oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion repre-
sent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients 
with SLE exhibit elevated 

Δ

  y   

m

 , i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alka-

linization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP. 
Increased production of nitric oxide has been identi fi ed as a cause of MHP and increased mitochondrial 
biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox- 
sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus 
T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a 
key sensor of MHP and mediator of enhanced Ca 

2+

   fl ux in lupus T cells.  

  Key words:

   Systemic lupus erythematosus ,  Mitochondrial hyperpolarization ,  Reactive oxygen inter-

mediates ,  Cytoplasmic alkalinization ,  Caspases ,  Glutathione depletion ,  ATP depletion ,  Apoptosis , 
 Necrosis ,  mTOR    

 

    Systemic lupus erythematosus (SLE) is a chronic in 

fl ammatory 

disease characterized by T and B cell dysfunction and production 
of antinuclear antibodies. Abnormal T cell activation and cell death 
underlie the pathology of SLE  

(  

1, 

  

2

  ) . Potentially autoreactive 

T and B lymphocytes during development  (  

3

  )  and after completion 

  1.   Introduction

62

A. Perl et al.

of an immune response are removed by apoptosis  (  

4

  ) . Paradoxically, 

lupus T cells exhibit both enhanced spontaneous apoptosis and 
defective activation-induced cell death. Increased spontaneous 
apoptosis of peripheral blood lymphocytes (PBL) has been linked 
to chronic lymphopenia  

(  

5

  )  and compartmentalized release of 

nuclear autoantigens in patients with SLE  (  

6

  ) . By contrast, defec-

tive CD3-mediated cell death may be responsible for persistence of 
autoreactive cells  (  

7

  ) 

  Both cell proliferation and apoptosis are energy-dependent pro-
cesses. Energy in the form of ATP is provided through glycolysis 
and oxidative phosphorylation. The mitochondrion, the site of 
oxidative phosphorylation, has long been identi fi ed as a source of 
energy and cell survival  (  

8

  ) . The synthesis of ATP is driven by an 

electrochemical gradient across the inner mitochondrial membrane 
maintained by an electron transport chain and the membrane 
potential (negative inside and positive outside). A small fraction of 
electrons react directly with oxygen and form reactive oxygen 
intermediates (ROI). Disruption of the mitochondrial membrane 
potential has been proposed as the point of no return in apoptotic 
signaling  (  

9–

  

11

  ) . Mitochondrial membrane permeability is subject 

to regulation by an oxidation–reduction equilibrium of ROI, pyri-
dine nucleotides (NADH/NAD 

NADPH/NADP) and GSH 

levels  (  

12

  ) . Regeneration of GSH by glutathione reductase from 

its oxidized form, GSSG, depends on NADPH produced by the 
pentose phosphate pathway (PPP)  (  

13

  ) . ROI levels and 

Δ

  y   

m

  are 

regulated by the supply of reducing equivalents from PPP  (  

14, 

  

15

  ) 

While ROI have been considered as toxic by-products of aerobic 
existence, evidence is now accumulating that controlled levels of 
ROI modulate various aspects of cellular function and are neces-
sary for signal-transduction pathways, including those mediating 
T cell activation and apoptosis  (  

16

  ) . 

 Increased production of ROI was demonstrated in TNF 

 (  

17–

  

19

  )  and Fas-mediated cell death  (  

9, 

  

14, 

  

20–

  

23

  ) . Disruption of 

the mitochondrial membrane potential (

Δ

  y   

m

 ) has been proposed 

as the point of no return in apoptotic signaling  (  

9–

  

11

  ) . Interestingly, 

elevation of 

Δ

  y   

m

 , mitochondrial hyperpolarization (MHP), and 

ROI production precede phosphatidylserine (PS) externalization 
and a disruption of 

Δ

  y   

m

  in Fas-  (  

15

  )  and H 

2

 O 

2

 -induced apoptosis 

of Jurkat human leukemia T cells and normal human peripheral 
blood lymphocytes  (  

24

  ) . These observations were extended to p53 

 (  

25

  ) , tumor necrosis factor  a   (  

26

  ) , staurosporin  (  

27

  ) , camptothe-

cin  (  

28

  ) , and nitric oxide-induced apoptosis  (  

29

  ) . Elevation of 

Δ

  y   

m

  

is independent from activation of caspases and represents an early 
event in apoptosis  (  

15, 

  

25

  ) . Pretreatment with caspase inhibitors, 

DEVD, Z-VAD, and Boc-Asp, completely abrogated Fas-induced 
PS externalization, indicating that activation of caspase-3, caspase-8, 

  1.1.   Mitochondrial 
Checkpoints of T Cell 
Activation and 
Apoptosis

63

4  Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients…

and related cysteine proteases were absolutely required for cell 
death  (  

30–

  

33

  ) . ROI levels were partially inhibited in DEVD-treated 

Jurkat cells, suggesting that caspase-3 activation, perhaps through 
damage of mitochondrial membrane integrity, contributes to ROI 
production and serves as a positive feedback loop at later stages of 
the apoptotic process. Nevertheless, ROI levels remained 
signi fi cantly elevated after pretreatment with caspase inhibitors. 
This suggested that activation of caspase-3 or caspase-8 was not 
required for increased ROI production and 

Δ

  y   

m

  hyperpolarization. 

By contrast, DEVD, Z-VAD, and Boc-Asp blocked PS externaliza-
tion and decline of 

Δ

  y   

m

  in annexin V-positive Jurkat cells, suggest-

ing that disruption of 

Δ

  y   

m

    (  

1

  )  was a relatively late event with 

respect to ROI production and 

Δ

  y   

m

  hyperpolarization and  (  

2

  )  

depended on activation of caspase-3 and related proteases. The 
precise mechanism by which Fas and TNF signaling leads to 
changes in 

Δ

  y   

m

  and ROI levels remains to be de fi ned. Cleavage of 

cytosolic bid by caspase-8 generates a p15 carboxyterminal frag-
ment that translocates to mitochondria. This may represent the 
initial insult to mitochondria in the Fas/TNF pathway  (  

34

  ) 

 MHP appears to be the earliest change associated with Fas 

 (  

15

  ) , H 

2

 O 

2

   (  

24

  ) , HIV-1  (  

35

  ) , p53  (  

25

  ) , TNF a   (  

26

  ) , staurosporin 

 (  

27

  ) , camptothecin  

(  

28

  ) , and NO-induced apoptosis  

(  

29

  ) 

Elevation of 

Δ

  y   

m

  is also triggered by activation of the CD3/CD28 

complex  (  

36

  )  or stimulation with Con A  (  

15

  ) , IL-10, IL-3, IFN- g , 

or TGF b   (  

37

  ) . Therefore, elevation of 

Δ

  y   

m

  or MHP represents an 

early but reversible switch not exclusively associated with apopto-
sis. With 

Δ

  y   

m

  hyperpolarization and extrusion of H+ ions from the 

mitochondrial matrix, the cytochromes within the electron trans-
port chain become more reduced which favors generation of ROI 
 (  

38

  ) . MHP is caused by exposure to nitric oxide (NO) which is 

produced during T cell activation  

(  

39

  ) . Reduced glutathione 

(GSH) is profoundly depleted in lymphocytes of SLE patients  (  

36

  )  

(Table  

1

 ), which may predispose to persistent MHP via 

 S -nitrosylation of complex I upon exposure to NO  (  

40

  ) . Thus, the 

effect of NO on MHP is tightly related to GSH levels. With MHP 
and extrusion of H+ ions from the mitochondrial matrix, the cyto-
chromes within the electron transport chain become more reduced 
which promotes ROI production and generates oxidative stress  (  

8

  ) 

Diminished production of GSH in face of MHP and increased 
ROI production are suggestive of a severe metabolic defect in 
lupus T cells. The activation of the mammalian target of rapamycin 
(mTOR) has recently emerged as a key sensor of MHP  (  

41

  )  and 

mediator of enhanced Ca 

2+

    fl ux in lupus T cells  

(  

42

  ) . mTOR 

activation may in 

fl uence Ca 

2+

    fl ux through interaction with 

HRES-1/Rab4, a regulator of endocytic recycling of CD3 and 
CD4 (Fig.  

1

 )  (  

43

  ) . 

   

64

A. Perl et al.

Fig. 1. (continued) space  (  

34

  ) . Phosphorylation of BAD by mitochondria-anchored PKA results in anti-apoptotic sequestration 

of BAD into the cytosol  (  

85

  ) . Signaling through cell death receptors, such as Fas  (  

15

  ) ,  CD3/CD28  co-stimulation   (  

36, 

  

37

  ) , 

ROS   (  

24

  ) ,  NO   (  

29

  ) , as well as lymphokines, IL-3, IL-10, IFN- g ,  and  TGF- b  

1

   in fl uence 

Δ

  y   

m

 , ATP synthesis and susceptibility 

to apoptosis  (  

37

  ) . MHP and mitochondrial biogenesis is mediated via production of NO by eNOS or nNOS  (  

39

  )   and 

 up-regulation of transcription factors PGC-1 a ,  Tfam,  and  ALAS   (  

86

  ) . NO production by eNOS may be compartmentalized to 

the T cell synapse  (  

87

  ) . NO causes transient MHP via reversible inhibition of complex IV/cytochrome c oxidase  (  

29

  )   and 

persistent MHP via  S -nitrosylation of complex I of the ETC in a state of GSH depletion  (  

40

  ) .  mTOR  senses 

Δ

  y   

m

    (  

41

  ) ,  interacts 

with the small GTPase HRES-1/Rab4  (  

43

  ) ,  and  regulates  Ca 

2+

   release   (  

42

  ).        

   Table  1 
  Signaling abnormalities of T cell death in patients with SLE   

 Signal 

 Effect 

 Reference 

 

Δ

  y   

m

  

 

 ROI 

, ATP 

 

  (  

36

  )  

 ROI 

 

 Spontaneous apoptosis 

, IL-10 production 

 

  (  

36, 

  

37

  )  

 GSH 

 

 ROI 

, Spontaneous apoptosis 

 

  (  

14, 

  

36

  )  

 Spontaneous apoptosis 

 

 Compartmentalized autoantigen release, disease 

activity 

 

  (  

5, 

  

6, 

  

36, 

  

76

  )  

 H 

2

 O 

2

  

 Apoptosis 

, necrosis 

 

  (  

36

  )  

 CD3/CD28  

AICD 

, necrosis 

 

  (  

37

  )  

 ATP 

 

 Predisposes for necrosis 

  (  

36, 

  

64

  )  

 Necrosis 

 

 In fl ammation 

 

  (  

36

  ) ,  (  

77

  )  

 AICD 

 

 Persistence of autoreactive cells 

  (  

7, 

  

37

  )  

 FasR 

 

 Spontaneous apoptosis 

 

  (  

76

  )  

 FasL 

 

 Spontaneous apoptosis 

 

  (  

47

  )  

 IL-10 

 

 Selective induction of apoptosis in SLE 

  (  

37, 

  

47, 

  

78

  )  

 NO 

 

 MHP, mTOR 

 

  (  

39, 

  

43, 

  

50

  )  

 IL-10 blockade 

 Spontaneous apoptosis 

, ROI 

 

  (  

37, 

  

47

  )  

 IL-12  

Spontaneous apoptosis 

, ROI 

 

  (  

37

  )  

  

: increase; 

: decrease  

  MHP predisposes for increased ROI production  (  

38

  ) . Oxidative 

stress affects activity of transcription factors AP-1 and NF- 

k

 B 

 (  

44, 

  

45

  ) , and, further downstream, may lead to the skewed 

expression of IL-2, TNF, and IL-10  (  

46

  ) . Increased spontaneous 

apoptosis of lymphocytes has been linked to increased IL-10 pro-
duction, release of Fas ligand, and overexpression of Fas receptor 
in SLE  (  

47

  ) . Since increased ROI levels confer sensitivity to H 

2

 O 

2

 , 

NO, TNF, and Fas-induced cell death  (  

14, 

  

15

  ) , elevated baseline 

  1.2.   Pharmacological 
Targeting 
of Mitochondrial 
Dysfunction in SLE

65

4  Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients…

  Fig. 1.    Overview of mitochondrial redox and metabolic checkpoints of T cell activation and apoptosis signals. Antigen 
binding-initiated signaling through the T cell receptor complex/CD3 and the CD28 co-stimulatory molecule activate phso-
phatidylinositol 3-kinase (PI3K) and protein tyrosine kinases (PTK). Increased cytosolic Ca 

2+

  concentration activates the 

serine/threonine phosphatase calcineurin which dephosphorylates the NFAT. Dephosphorylated NFAT can translocate to the 
nucleus where it promotes transcription of IL-2 in concert with AP-1, NF6B, and Oct-1. Ca 

2+

   fl ux into mitochondria increases 

production of ROS and NF-6B activation  (  

79–

  

81

  ) . Mitochondrial membrane integrity is maintained by a balance of mem-

brane-stabilizing bcl-2 and bcl-X 

L

  and pore-inducing bax and bad  (  

34

  )  as well as the metabolic capacity to synthesize 

reducing equivalents, NADPH, GSH, and TRX. Controlled increase of ROS levels activates NF-6B and promotes cell growth. 
Excess ROS production and disruption of 

Δ

  y   

m

  lead to activation-induced cell death executed by caspase 3 (digesting vitally 

important proteins PARP, 70K U1RNP, lamin, and actin) and caspase 3-dependent DNase (CAD, causing nuclear DNA frag-
mentation). Cleavage by caspase 3 is thought to expose cryptic epitomes and cause autoantigenicity of self antigens  (  

82

  ) . 

Activity of redox-sensitive transcription factors NF-6B, p53, AP-1, and Sp1 is regulated through release from inhibitor 
complexes and conformational changes in their active sites. Intracellular antioxidants reduced glutathione (GSH) and thi-
oredoxin (TRX-DT) are regenerated at the expense of NADPH supplied primarily through metabolism of glucose via the 
pentose phosphate pathway (PPP)  (  

83

  ) . Among PPP products, ribose 5-phosphate is required for nucleotide and DNA syn-

thesis and support cell growth, C3–C7 sugars in fl uence mitochondrial function and ROS production, inositol and ADP-ribose 
serve as precursors for second messengers, inositol phosphates and cADP-ribose, respectively. Dehydroascorbate (DHA) is 
imported through GLUT1. DHA is metabolized through the PPP, thereby enhancing GSH levels. DHA also increases surface 
expression of Fas-R  (  

84

  ) . Glutathione reductase and TRX reductase synthesize GSH and TRX-DT at the expense of NADPH. 

Formulation of the PPP and its ef fi ciency to provide NADPH is dependent on the expression of G6PD and TAL  (  

14, 

  

15

  ) . 

Δ

  y   

m

  

is controlled by intracellular GSH/NADH/NADPH levels, integrity of the permeability transition pore complex largely com-
prised of adenine nucleotide translocator (ANT, inner membrane), voltage-dependent anion channel (VDAC, outer mem-
brane), and translocation and dimerization of pro- and anti-apoptotic bcl-2 family members in the intermembrane 

 

66

A. Perl et al.

Δ

  y   

m

 , ROI production, and pH 

i

  may have key roles in altered 

activation and death of lupus T cells. Although MHP was not 
affected, IL-10 antibody or IL-12 normalized ROI production 
and intracellular alkalinization in lupus PBL  (  

37

  ) . Therefore, IL-10 

antagonists may partially correct signaling dysfunction in lupus. 

 Recent studies showed diminished GSH/GSSG ratios in the 

kidneys of 8-month-old vs. 4-month-old (NZB × NZW) F1 mice; 
treatment with  N -acetylcysteine (NAC), a precursor of GSH and 
stimulator of its de novo biosynthesis, prevented the decline of 
GSH/GSSG ratios, reduced autoantibody production and devel-
opment of glomerulopnephritis (GN) and prolonged the survival 
of (NZB × NZW) F1 mice  (  

48

  ) . Oral NAC has been used to treat 

oxidative stress in patients with idiopathic pulmonary  fi brosis (IPF) 
 (  

49

  ) . In a 1-year study of IPF patients treated with prednisone and 

azathioprine, addition of NAC (3 × 600 mg/day) improved vital 
capacity and reduced myelotoxicity in comparison to placebo. 
Therefore, prospective clinical studies appear justi 

fi ed to assess 

whether NAC treatment can reverse GSH depletion, correct T cell 
signaling defects and provide clinical bene 

fi t to patients with 

lupus. 

 NO production is a particularly interesting target because it 

provides a link between seemingly dissociated features of T cell 
activation and mitochondrial function. NO induces MHP and 
mitochondrial biogenesis, increases Ca 

2+

  in the cytosol and mito-

chondria of normal T cells, and recapitulates the enhanced CD3/
CD28-induced Ca 

2+

   fl uxing of lupus T cells  (  

50

  ) . NO contributes 

to the development of GN in the MRL/ lpr  lupus mouse model 
 (  

51

  ) . Inactivation of iNOS does not block the development of 

lupus  

(  

52

  ) , suggesting a role for eNOS and nNOS isoforms 

expressed in T cells. However, given the widespread expression of 
these isoforms in vascular smooth muscle and brain, it will be nec-
essary to develop T-cell-speci fi c approaches for inhibiting NOS to 
avoid potentially deleterious side effects.   

 

      1.    Ficoll-Paque Plus (Amersham-Pharmacia, Uppsala, Sweden).  

     2.    RPMI 1640 medium, fetal calf serum, penicillin, streptomycin, 

amphotericin B (Life Technologies, Grand Island, NY).  

     3.    OKT3 monoclonal antibody (CRL 8001 from ATCC, 

Rockville, MD).  

     4.    CD28.2 monoclonal antibody (Pharmingen, San Diego, CA).  

     5.    Cytokines: IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, 

TNF- a , TGF- b  

1

 , and IFN- g  (PeproTech, Rocky Hill, NJ).  

  2.   Materials

67

4  Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients…

     6.    Polyclonal goat anti-human IL-10 neutralizing antibody (R&D 

Systems, Minneapolis, MN).  

     7.    Annexin binding buffer: 10 mM HEPES pH 7.4, 140 mM 

NaCl, and 2.5 mM CaCl 

2

 .  

     8.    Phosphate-buffered saline (PBS): 8 g of NaCl, 0.2 g of KCl, 

1.44 g of Na 

2

 HPO 

4

 , 0.24 g of KH 

2

 PO 

4

  dissolved in 1 l of H 

2

 O 

with pH adjusted to 7.4.  

     9.    Fluorescein-conjugated annexin V (annexin V-FITC) and phy-

coerythrin-conjugated annexin V (annexin V-PE, R & D 
Systems, Minneapolis, MN).  

    10.    Propidium iodide (R&D Systems).  

    11.    Triton X-100 (Sigma, St. Louis, MO).  

    12.    Hydroethidine (HE, Molecular Probes, Eugene, OR).  

    13.    Quantum Red/Cy5-conjugated monoclonal antibodies 

directed to CD3, CD4, CD8, CD14 (Sigma, St. Louis, MO), 
CD45RA, and CD45RO (Pharmingen, San Diego, CA).  

    14.    Fluorescence microscope: Nikon Eclipse E800 camera (Nikon 

Corporation, Tokyo, Japan). Equipped with SPOT digital 
camera (Diagnostic Instruments, Sterling Heights, MI).  

    15.    Flow cytometer: Becton Dickinson FACStar Plus  fl ow cytom-

eter equipped with an argon ion laser delivering 200 mW of 
power at 488 nm.  

    16.    Oxidation-sensitive  

fl uorescent probes 5,6-carboxy-2 

¢

 ,7 ¢ -

dichloro fl uorescein-diacetate (DCFH-DA), dihydrorhodamine 
123 (DHR) and hydroethidine (HE, Molecular Probes, 
Eugene, OR).  

    17.    Cationic lipophilic dyes with high binding af 

fi nity to 

mitochondria: 3,3 

¢

 -dihexyloxacarbocyanine iodide 

(DiOC 

6

 ), 5,5 

¢

 ,6,6 ¢ -tetrachloro-1,1 ¢ ,3,3 ¢ -tetraethylbenzimida

zolocarbocyanine iodide (JC-1), tetramethylrhodamine, 
methyl ester, perchlorate (TMRM), all from Molecular Probes 
(Eugene, OR).  

    18.    Carbonyl cyanide  

m -chlorophenylhydrazone (mClCCP, 

Sigma).  

    19.    Luminometer: AutoLumat LB953 (Berthold GmbH, Wildbad, 

Germany).  

    20.    ATP determination kit (Molecular Probes, Eugene, OR).  

    21.    ApoGlow kit (Lumitech, Nottingham, UK).  

    22.    Carboxy SNARF-1-acetoxymethyl ester acetate (SNARF-1, 

Molecular Probes, Eugene, OR) 23. DMSO (Sigma).  

    23.    High K+ buffers of varying pH values (120 mM KCl, 30 mM 

NaCl, 0.5 mM MgSO 

4

 , 1 mM CaCl 

2

 , 1 mM NaHPO 

4,

  5 mM 

glucose and 10 mM HEPES).  

68

A. Perl et al.

    24.    Nigericin (Sigma, St. Louis, MO; diluted from a stock solution 

of 500  m g/ml in ethanol).  

    25.    Deproteinizing buffer for glutathione (GSH) assay: 70 % per-

chloric acid and 15 mM bathophenanthrolinedisulfonic acid 
(BPDS, Sigma).  

    26.     g -Glutamyl glutamate ( g -Glu-Glu, Sigma), internal standard 

for GSH assay.  

    27.    After repeated freezing and thawing, samples were centrifuged 

at  15,000 ×  g  for 3 min. 50  m l of 100 mM mono-iodo-acetic 
acid in 0.2 mM  m -cresol purple was added to 500  m l superna-
tant. Samples were neutralized by addition of 480  m l of 2 M 
KOH and 2.4 M KHCO 

3

  and incubated in the dark at room 

temperature for 10 min. Then, 1 ml of 1 %  fl uoro-dinitro-
benzene was added and the samples were incubated in the dark 
at 4 °C overnight. After centrifugation and  fi ltering, 100  m l of 
supernatants were injected into the HPLC Model 2690 (Waters 
Alliance System, Milford, MA) equipped with a Model 996 
photodiode array detector and Spherisorb NH 

2

  column 

(4.6 × 250 mm; 10  m m; Waters).  

    28.    7-Amino-4-tri fl uoromethyl-coumarin (AFC, Sigma).  

    29.    Caspase substrate peptides: DEVD-AFC, Z-IETD-AFC, where 

Z represents a benzyloxycarbonyl group; caspase inhibitor 
peptides Z-Val-Ala-Asp(Ome).fmk (Z-VAD), Boc-Asp.fmk 
(Boc-Asp) as well as non-caspase cysteine protease inhibitor, 
Z-Phe-Ala.fmk (Z-FA) can be obtained from Enzyme Systems 
Products (Livermore, CA).  

    30.    Caspase assay buffer: 250 mM sucrose, 20 mM HEPES–KOH 

pH 7.5, 50 mM KCl, 2.5 mM MgCl 

2

 , 1 mM dithiothreitol.  

    31.    Versene (Life Technologies).  

    32.    Concanavalin A (Con A, Sigma).  

    33.    Goat anti-mouse IgG (ICN, Aurora OH).  

    34.    Tritiated thymidine,  

3

 HTdR (ICN).  

    35.    CH-11 IgM monoclonal antibody to Fas/Apo-1/CD95 

(Upstate Biotechnology, Saranac Lake, NY).  

    36.    Complete RPMI medium: RPMI 1640 supplemented with 

10 % fetal calf serum, 2 mM  

L

 -glutamine, 100 IU/ml penicil-

lin, 100  m g/ml gentamicin, and 10  m g/ml amphotericin B.  

    37.    Plastic tissue culture dishes (Becton Dickinson, Franklin 

Lake, NJ).  

    38.    Phenol, chloroform, isoamyl alcohol, proteinase K, agarose 

(all RNase and DNase-free. molecular biology grade, from 
Sigma).  

    39.    Spectrophotometer.  

69

4  Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients…

    40.    Luminometer.  

    41.    Carbonyl cyanide  

m -chlorophenylhydrazone (mClCCP, 

Sigma).  

    42.    Folin & Ciocalteu’s Phenol Reagent Solution (Sigma).  

    43.    4 mm diameter 0.45  

m

 m polypropylene  

fi lter (Whatman, 

Mainstead, England).  

    44.    Monoclonal antibody to poly(ADP-ribose) polymerase (PARP) 

C-2-10  (  

53

  ) .  

    45.    Monoclonal antibody 5F7 directed to C-terminal amino acids 

176-460 of human FLICE/Mch5/caspase-8 (Panvera, 
Madison, WI).  

    46.    Monoclonal antibody 31A1067 directed to caspase 3 (Gene 

Therapy Systems, San Diego, CA).  

    47.    Monoclonal antibody C4 directed to human  

b

  actin 

(Boehringer, Indianapolis, IN).  

    48.    Biotinylated secondary antibodies and horseradish peroxidase-

conjugated avidin (Jackson Laboratories, West Grove, PA).  

    49.    4-Chloronaphthol (Sigma).  

    50.    Enhanced chemiluminescence detection kit (Western Lightning 

Chemiluminescence Reagent Plus, PerkinElmer Life Sciences, 
Boston, MA).  

    51.    Kodak Image Station 440CF equipped with Kodak 1D 

Image Analysis Software (Eastman Kodak Company, 
Rochester, NY).      

 

 The methods described below outline (1) in vitro lymphocyte cul-
ture, activation, and apoptosis assays,  fl ow cytometric analysis of 
(2) 

Δ

  y   

m

  and (3) ROI production and (4) intracellular pH, (5) 

measurement of intracellular ATP and ADP, (6) HPLC analysis of 
reduced (GSH) and oxidized forms of glutathione (GSSG), and 
(7) caspase enzyme assays (see Note 1). 

        1.    Collect peripheral blood in sterile tubes containing 50 U hepa-

rin (Sigma) per ml of blood.  

     2.    Layer blood diluted 1:1 with PBS on Ficoll-Paque. Typically 

layer 10 ml diluted blood over 5 ml of Ficoll-Paque.  

     3.    Centrifuge cells at 500 

× 

 

g  for 30 min with centrifuge 

brake off.  

  3.   Methods

  3.1.   Lymphocyte 
Culture, Activation, 
and Viability Assays

  3.1.1.   Separation 

of Peripheral Blood 

Mononuclear Cells

70

A. Perl et al.

     4.    Remove peripheral blood mononuclear cells (PBMC) from 

interface between Ficoll-Paque and plasma with pipettor.  

     5.    Wash PBMC three times in PBS by centrifugation at 300 ×  g  

for 10 min.  

     6.    PBMC are resuspended at 10 

6

  cells/ml in RPMI 1640 medium, 

supplemented with 10 % fetal calf serum, 2 mM  

L

 -glutamine, 

100 IU/ml penicillin, and 100  m g/ml gentamicin (complete 
RPMI medium) and incubated for experiments at 37 °C in a 
humidi fi ed atmosphere with 5 % CO 

2

 .      

       1.    Precoat Petri dishes with autologous serum for 30 min 

at 37 °C.  

     2.    Add 5 ml of PBMC (maximum 5 × 10 

6

 /ml) to serum-pre-

treated dishes and incubate for 1 h at 37 °C.  

     3.    Remove nonadherent cells by washing three times with 5 ml of 

warm (37 °C) complete RPMI medium.  

     4.    To obtain a monocyte-enriched cell fraction wash dishes vigor-

ously with warm medium.  

     5.    Add 4 ml of ice-cold 0.05 % Versene and 1 ml autologous 

serum to each dish for 15 min at room temperature.  

     6.    Scrape off loosely adherent monocytes with a rubber police-

man under inverse microscopic control.  

     7.    The monocyte-depleted fraction of peripheral blood lympho-

cytes (PBL) should contain less than 2 % monocytes, while the 
monocyte-enriched fraction should contain 90–95 % mono-
cytes by staining with CD14 monoclonal antibody.      

  Human PBL undergo apoptosis in response to repetitive activation 
through the T cell receptor, i.e., CD3/CD28 co-stimulation 
resulting in activation-induced cell death (AICD)  (  

36, 

  

37

  ) , cross-

linking of cell surface death receptors such as Fas/Apo-1/CD95 
 (  

15

  )  or elevation of intracellular ROI levels after treatment with 

2

 O 

2

   (  

24, 

  

36

  ) . Monocytes/macrophages remove apoptotic bod-

ies via phagocytosis, therefore processing of cell death signals by 
lymphocytes is best evaluated using PBL (see Note 2). 

       1.    Precoat 10 cm diameter plastic Petri dishes with 100  m g/ml 

goat anti-mouse IgG (diluted in PBS) for 2 h at 37 °C.  

     2.    Wash plates with PBS, add OKT3 monoclonal antibody (1  m g/

ml), and incubate for 1 h at 37 °C.  

     3.    Add PBL (10 

6

  cells/ml in complete RPMI medium).  

     4.    For CD28 co-stimulation add 500 ng/ml mAb CD28.2 and 

incubate cells at 37 °C for the desired period of time.      

  3.1.2.   Separation 

of Monocytes and 

Peripheral Blood 

Lymphocytes

  3.1.3.   Cell  Culture, 

Activation, and Viability 

Assays

   CD3/CD28  Co-stimulation 

of PBL