Use of flow cytometry and monochlorobimane to quantitate

intracellular glutathione concentrations in feline leukocytes

Craig Webb a, Cathy Bedwell b, Amanda Guth b, Paul Avery b, Steven Dow a,b,*

a Department of Clinical Sciences, Colorado State University, Ft. Collins, CO 80523, United Statesb Department of Microbiology, Immunology, and Pathology, Colorado State University, Ft. Collins, CO 80523, United States

Received 6 December 2005; received in revised form 31 January 2006; accepted 13 February 2006

Abstract

Oxidative stress and abnormal glutathione metabolism is thought to play an important role in various diseases of cats.

However, current assays for the reduced form of glutathione (GSH) are time-consuming and semi-quantitative and do not allow

assessment of GSH concentrations in individual cell populations. Therefore, we developed a flow cytometric assay for rapid

determination of intracellular GSH concentrations in feline blood leukocytes. The assay was based on the ability of the non-

fluorescent substrate monochlorobimane (mBCl) to form fluorescent adducts with GSH in a reaction catalyzed by the enzyme

glutathione-S-transferase. Using flow cytometry, we found that mBCl was sensitive and specific for intracellular detection of the

reduced form of GSH in feline leukocytes. Intracellular GSH concentrations were also stable for at least 24 h in EDTA preserved

whole blood samples stored at 4 8C. Neutrophils and monocytes from normal cats had significantly higher intracellular

concentrations of GSH than T cells and B cells. The effects of FIV infection on intracellular GSH concentrations in cats were

assessed using flow cytometry. We found that neutrophils from FIV-infected cats had significantly increased GSH concentra-

tions, whereas intracellular GSH concentrations were significantly decreased in CD4+ and CD8+ lymphocytes from FIV-infected

cats, compared to age-matched control animals. We conclude that a flow cytometric assay based on mBCl may be used to

accurately and rapidly assess the effects of various disease states and treatments on GSH concentration in cat leukocytes and to

help assess intracellular oxidative stress.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Cat; Leukocyte; Glutathione; FIV; Monochlorobimane

www.elsevier.com/locate/vetimm

Veterinary Immunology and Immunopathology 112 (2006) 129–140

1. Introduction

Glutathione plays a key role in the maintenance of

intracellular oxidative balance in all cells in the body

* Corresponding author. Tel.: +1 970 491 6144;

fax: +1 970 491 0603.

E-mail address: sdow@colostate.edu (S. Dow).

0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved

doi:10.1016/j.vetimm.2006.02.009

(Droge, 2002). The reduced form of glutathione (GSH)

provides a large portion of the intracellular reducing

power available to the cell and therefore determines to a

large degree the cell’s ability to eliminate potentially

harmful free radicals, reactive oxygen species and

metabolic by-products (Mytilineou et al., 2002).

Changes in GSH concentration and the intracellular

redox state of the cell have also been shown to affect

.

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140130

intracellular signal transduction, gene expression,

cytokine production, and induction of apoptosis

(Finkel, 2003; Nathan, 2003). Given the key role that

GSH plays in regulating oxidative balance, it is not

surprising that reductions in intracellular concentra-

tions of GSH, which is associated with oxidative stress,

play an important role in a variety of diseases. For

example, diseases previously associated with abnormal

intracellular GSH concentrations in humans include

HIV infection, diabetes mellitus, renal failure, liver

disease, neurologic disorders, and neoplasia (Aukrust

and Muller, 1999; Aukrust et al., 2003; Kiessling et al.,

1999; Siems et al., 2002; Staal, 1998).

Currently, most assays of GSH concentrations rely

on tests that measure total glutathione concentration

(reduced and non-reduced) in lysates of whole blood

or tissues. These assays are time-consuming to run and

do not provide information on the concentration of

GSH in individual populations of cells (Richie et al.,

1996). Since intracellular concentrations of GSH often

vary widely from one cell type to another, the results

of whole blood or tissue lysate assays can be

influenced significantly by changes in the relative

proportion of different cell types present in these

tissues (Scott et al., 1990; Shrieve et al., 1988).

Therefore, an assay capable of accurately assessing

GSH concentrations on a per cell basis could greatly

increase the amount of information gathered from

analyzing blood or tissue samples.

An assay based on flow cytometry offers the greatest

potential for analysis of GSH concentrations in

individual cells. Such assays have been used previously

to assess oxidative balance in cells from humans. For

example, flow cytometry was used to analyze GSH

content in leukocytes from blood samples of humans

(Hedley et al., 1990; Scott et al., 1990). Several different

non-fluorescent halogenated bimanes have been eval-

uated for use in detecting and quantitating intracellular

GSH concentration in leukocytes. The most widely

used bimanes for this application are monobromobi-

mane (mBBr) and monochlorobimane (mBCl). The

reaction between mBCl and GSH is catalyzed

specifically by the enzyme glutathione-S-transferase

(GST), whereas the reaction catalyzed by mBBr is not

specifically dependent on GST. The enzymatic reaction

with GSTis therefore thought to confer specificity to the

mBCl reaction for detection of intracellular GSH

(Hedley and Chow, 1994). However, studies suggest

that while mBCl is specific for detection of GSH in rat

cells, it is not specific for quantitating GSH in human

cells, due to species differences in GST isoenzymes

(Cook et al., 1991).

Flow cytometry has been used to evaluate

leukocyte subsets in cats (Byrne et al., 2000), but

the use of flow cytometry to assess intracellular GSH

content in cats has not been reported previously. Such

an assay would be particularly useful for studies in

cats, given their unique sensitivity to oxidative stresses

(Fettman et al., 1999). Therefore, we conducted

studies to develop a flow cytometric assay for

determination of intracellular GSH concentrations

in blood samples from cats. The utility of such a flow

cytometric assay was evaluated in a study of the

effects of FIV infection on intracellular GSH

concentrations in leukocytes of cats.

2. Materials and methods

2.1. Blood samples from healthy cats

Blood samples from 32 healthy cats with normal

complete blood count (CBC) results were obtained

from client-owned cats presented to the Colorado

State University Veterinary Teaching Hospital (CSU-

VTH) for routine wellness examinations. All healthy

cats used for evaluating GSH assays were also normal

on physical examination and none of the animals were

anemic. Protocols for these studies were approved by

the Animal Care and Use Committee at Colorado State

University.

2.2. FIV infection of SPF cats

Nine, 16-week-old cats from a specific pathogen-

free colony maintained at the Colorado State University

were inoculated s.c. with 1 � 108 viral copies in 1 ml of

cell-free pooled plasma from cats infected with an FIV

clade B virus (Dow et al., 1999; O’Neil et al., 1996).

Infection was confirmed by serologic detection and by

PCR detection of FIV provirus within 1 month of

inoculation (data not shown). Five healthy age-matched

cats from the same cat colony served as uninfected

controls. Twelve weeks after FIV inoculation, blood

samples were collected from each of the infected and

control cats and intracellular GSH concentrations were

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140 131

analyzed. A CBC was also performed on each cat at the

same time. These studies were approved by the Animal

Care and Use Committee at Colorado State University.

2.3. Preparation of blood samples for flow

cytometry

Peripheral blood samples were obtained by jugular

venipuncture and preserved in EDTA tubes and stored

at 4 8C and processed for analysis within 6 h of

acquisition, unless otherwise noted. Blood for analysis

was processed to remove RBC. Briefly, 200 ml of

EDTA-preserved blood was added to 15 ml of NH4Cl

erythrocyte lysis buffer and incubated for 15 min at

room temperature. The sample was then washed twice

in HBSS solution (Sigma–Aldrich, St. Louis, MO) and

the leukocytes were resuspended in 500 ml of FACS

buffer (PBS with 2% FBS and 0.1% sodium azide) and

stored at 4 8C for less than 30 min prior to analysis. This

procedure typically yielded a final cell concentration in

the range of 4 � 106 cells/ml. Resuspending leukocyte

samples in FACS buffer for <30 min did not affect

intracellular GSH concentrations (data not shown).

2.4. Use of monochlorobimane (mBCl) for

detection of intracellular GSH concentration

Monochlorobimane (Molecular Probes, Eugene,

OR) was dissolved in 100% ethanol to a stock

concentration of 40 mM and stored at�20 8C. Special

precautions were used to minimize the exposure of

mBCl to ambient light. Monochlorobimane was added

to the leukocyte suspension to a final concentration of

40 mM and the cells were maintained at room

temperature in the dark for 20 min prior to analysis

of the cells. Monobromobimane (mBBr) was prepared

and stored exactly as described for mBCl.

2.5. Determination of specificity of mBCl for

detection of intracellular GSH

To assess and compare the specificity of mBCl and

mBBr for detection of intracellular GSH, leukocytes

were incubated with N-ethylmaleimide (NEM;

Sigma), a GSH depleting agent, which has been used

previously to establish the specificity of mBCl for

detection of GSH in human leukocytes (Hedley and

Chow, 1994). N-ethylmaleimide was prepared as a

stock solution in 100% ethanol and was added to

suspensions of feline leukocytes to a final concentra-

tion of 100 mM for 10 min at room temperature prior

to addition of mBBr or mBCl. In other experiments,

serial log dilutions of NEM were added to leukocytes

prior to addition of mBCl or mBBr.

2.6. Spectrophotometric determination of GSH

concentration in blood

A commercial assay for determination of GSH

content in whole blood (Bioxytech GSH-400 kit,

OXIS Research, Portland, OR) was used to assess

GSH concentrations in unseparated leukocytes. The

assay was performed according to manufacturer’s

directions and has been used previously to quantitate

GSH concentrations in blood and tissue samples from

cats (Center et al., 2005, 2002).

2.7. Chromatographic determination of mBCl

specificity for GSH

For confirmation of the specificity of mBCl for GSH

in vitro, a high performance liquid chromatographic

system was utilized, which consisted of Waters

Baseline 810 software, a Waters 501 HPLC pump, a

Waters 700 Satellite WISP autosampler, a Waters Nova-

Pak 8NVC18 4m radial compression column (Millipore

Corporation, Miliford, MA) and a Shimadzu RF-535

fluorescence detector (Shimadzu Corporation, Kyoto,

Japan). The excitation wavelength of the fluorescence

detector was set at 394 nm and emission wavelength

was set at 490 nm. The eluent, 80% methanol 20%

water, was pumped through the column at 1 ml/min. A

10 ml injection volume was utilized and data were

collected for 10 min at a rate of 1 data point per second.

Purified GSH and glutathione-S-transferase were

purchased from Sigma–Aldrich (St. Louis, MO) and

mBCl was purchased from Molecular Probes.

2.8. Determination of leukocyte GSH

concentrations using flow cytometry

Intracellular GSH concentrations were determined

by flow cytometry using a Cyan MLE flow cytometer

(DakoCytomation, Ft. Collins, CO). Prior to each day’s

experiments, the flow cytometer was calibrated using

SpectraAlign flow cytometry beads (DakoCytomation)

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140132

to assure consistent MFI readings between different

experiments. Monochlorobimane fluorescence was

assessed using a UV laser with excitation wavelength

at 350 nm. Dead cells were excluded from analysis and

monocytes, lymphocytes, and neutrophils were identi-

fied by their distinct forward-angle versus side-angle

light scatter characteristics. For most experiments,

lymphocytes, monocytes, and neutrophils were gated

based on specific forward and side-scatter character-

istics (Byrne et al., 2000). In some experiments,

antibodies specific for feline leukocyte cell surface

determinants were used to confirm the accuracy of

gating (see below). Data were analyzed using Summit1

software (DakoCytomation).

For assessment of GSH concentration in specific

cell populations, immunostaining was done using the

following antibodies: anti-feline CD4-fitc (clone vpg

34; Serotec), anti-feline CD8-pe (clone vpg 9;

Serotec) (Dean et al., 1991), a cross-reactive antibody

to CD14 (clone Tuk4; pe-cy5 conjugated; Serotec)

(Willett et al., 2003) and a cross-reactive antibody to

B220 (CD45R) (clone RA3-6B2; biotin-conjugated,

eBioscience). Co-staining experiments with the B220

antibody (eBioscience) and an anti-B canine B cell

antibody (Serotec) produced staining of the same

population of cells; staining with the B220 antibody

and an anti-human CD19 antibody also produced co-

staining of nearly the same population of feline

peripheral blood mononuclear cells (Dr. Anne Avery,

Colorado State University; personal communication).

Samples were incubated with normal cat serum to

block non-specific binding, then washed and incu-

bated with the appropriate antibody combinations for

20 min at 4 8C. Immediately after cell surface

staining, samples were treated with mBCl as described

above and analyzed by flow cytometry.

2.9. Statistical analyses

Comparisons between two samples for determina-

tion of statistically significant differences were done

using Student’s t-test. For comparisons between three

or more treatment groups, ANOVA was done,

followed by Tukey’s multiple means comparison test.

Statistical analyses were done using GraphPad Prism

software (San Diego, CA). A p-value < 0.05 was

considered significant for all statistical analyses

performed in this study.

3. Results

3.1. Assessment of the specificity of

monochlorobimane for detection of intracellular

GSH in feline leukocytes

It was first determined that mBCl could in fact react

with intracellular thiols in feline leukocytes and that

this response could be detected using flow cytometry.

Feline peripheral blood leukocytes from healthy

client-owned cats were reacted with 40 mM mBCl

for 20 min, then the samples were analyzed by a flow

cytometer equipped with a 350 nm UV laser. Typical

flow cytometry results for neutrophils incubated with

mBCl are shown in Fig. 1A. The mean fluorescence

intensity (MFI) for neutrophils excited with a 350 nm

laser increased from 5 (open histogram) to 236 (filled

histogram) following incubation with mBCl. Thus,

mBCl was an efficient means of detecting intracellular

thiols in feline leukocytes. The ability of a different

bimane (monobromobimane; mBBr) to react with

intracellular thiols in feline leukocytes was also

assessed. After incubation with 40 mM mBBr for

20 min, neutrophil fluorescence was measured. We

found that incubation with mBBr also produced a

large shift in fluorescence intensity, from an MFI of 5

in non-treated samples to an MFI of 354 following

incubation with mBBr (data not shown). Therefore,

both mBCl and mBBr efficiently labeled intracellular

thiols in cat leukocytes.

Experiments were conducted next to assess the

specificity of the labeling reactions with mBCl and

mBBr. Leukocytes were pre-incubated with the GSH

depleting agent N-ethylmaleimide (NEM). Following

a 10 min incubation with 100 mM NEM, cells were

incubated with mBCl or mBBr and the cells analyzed

by flow cytometry. The fluorescence intensity was

then compared between NEM-treated and untreated

cells. We found that pre-treatment with NEM

markedly reduced the fluorescence intensity of feline

neutrophils (Fig. 1B) and feline lymphocytes (data not

shown). In Fig. 1A, a typical 350 nm fluorescence

histogram for neutrophils incubated with (hatched) or

without NEM (filled) and then treated with mBCl is

shown. Treatment with NEM reduced the MFI of

neutrophils treated with mBCl by over 96%, from 236

to 9. The NEM competition experiments were

repeated using triplicate samples of cat neutrophils

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140 133

Fig. 1. Assessment of the specificity of mBCl for quantitation of

intracellular GSH in cat leukocytes. Leukocytes were prepared from

peripheral blood of normal cats and analyzed for detection of

intracellular GSH, as described in Section 2. (A) Representative

histogram of neutrophils, either untreated (open histogram;

MFI = 5), treated with mBCl (filled histogram; MFI = 236), or

pre-treated with 100 mM N-ethylmaleimide (NEM) for 10 min,

and then treated with mBCl (hatched histogram; MFI = 9) and

analyzed by flow cytometry as described in Section 2. (B) Average

(�S.D.) mean fluorescence intensity at an emission wavelength of

350 nm (MFI 350 nm) of neutrophils from six healthy control cats

that were untreated, treated with mBCl only (see above), or pre-

treated with NEM and then treated with mBCl. * denotes significant

differences ( p < 0.001), as assessed by ANOVA and Tukey’s multi-

ple means comparison. (C) Effects of serial dilutions of NEM on

neutrophil GSH concentration in six healthy cats, as determined by

mBCl and flow cytometry. Similar results were obtained in one

additional experiment.

from six normal cats and the overall effect of NEM

pre-treatment was determined. The mean (�S.D.) MFI

for untreated cells was 5.7 � 0.7 and for mBCl treated

cells it was 172 � 13. For samples pre-incubated with

NEM prior to mBCl loading, the mean MFI was

9.0 � 0.7. Pre-treatment with NEM produced a highly

significant ( p < 0.001) reduction in mBCl emission

compared to pre-treated samples (Fig. 1B). The MFI

of NEM pre-treated samples was not statistically

different ( p > 0.05) from that of samples not treated

with mBCl. A dose–response curve for use of NEM

for competition with mBCl for intracellular GSH was

also determined (Fig. 1C). These studies indicated that

pre-treatment with 100 mM NEM produced almost

complete inhibition of mBCl fluorescence, while

inhibition by NEM was still detected following

incubation with as little as 100 nM NEM.

Next, the usefulness of mBBr for measuring

intracellular GSH in cats was assessed. When NEM

competition experiments were repeated using mBBr

as the substrate, we found that NEM pre-treatment

resulted in only a 90% inhibition of the mBBr-elicited

fluorescence (data not shown).

3.2. Chromatographic confirmation of mBCl

specificity for GSH as conferred by the GST

enzyme

The specificity of mBCl for the GST catalyzed

reaction with GSH was assessed chromatographically,

using reagent grade GSH and GST (Sigma) and mBCl

(Molecular Probes). Standard concentrations of GSH

(5, 25, and 50 mM) were prepared in PBS and then

labeled with 20 mM mBCl, in the presence or absence

of 1 unit of GST per mM GSH. We found that the peak

area of the 50 mM GSH standard prepared with GST

(341075) was nearly 50 times greater than that of the

standard prepared without GST (6953). These results

indicated that the reaction of mBCl with GSH was

highly dependent on the presence of GST.

3.3. Combined flow cytometry and

spectrophotometry for assessment of GSH content

in feline leukocytes

An experiment was conducted to quantitate

intracellular GSH concentrations in different leuko-

cyte subpopulations using the mean fluorescence

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140134

intensity (MFI) data generated by the mBCl flow

cytometric assay. One aliquot of 2 � 106 leukocytes

from a normal cat (with RBCs removed by lysis) was

analyzed for GSH content using a commercially

available colorimetric method (Bioxytech GSH-400

kit, OXIS Research, Portland OR). Briefly, the GSH

content of the leukocyte sample was determined using

a spectrophotometer by comparing the sample

absorbance to that of a standard curve generated

using known concentrations of GSH. The MFI of a

duplicate aliquot of 2 � 106 leukocytes from the same

cat was also determined by flow cytometry using

mBCl. By this approach, we obtained a value for both

the MFI and the GSH concentration for the total

leukocyte sample. By applying the appropriate gating

paradigm to the mBCl-treated sample, we determined

the absolute number of neutrophils, monocytes and

lymphocytes, as well as the MFI for each individual

cell population. Using this data, we calculated the

approximate GSH content of the individual cell

populations by comparison with the overall leukocyte

GSH content that was determined using the commer-

cial GSH assay. This approach is similar to the one

used previously to determine GSH content in human

leukocytes and assumes a linear relationship between

MFI and GSH content in leukocytes (Scott et al.,

1990). We found that the GSH content of cat

neutrophils was 37 nmol/107 cells, while the content

in monocytes was 28 nmol/107 cells, and the content

in lymphocytes was 7 nmol/107 cells. These values

were comparable to those determined previously in

human leukocytes. For example, it has been reported

previously that human leukocytes GSH concentrations

were 12.5 nmol per 107 neutrophils, 14.5 nmol per 107

monocytes, and 5.0 nmol per 107 lymphocytes (Scott

et al., 1990). Moreover, using flow cytometry and

mBCl we determined the MFI generated by a known

number of erythrocytes and calculated that feline

erythrocytes contained approximately 1.0 nmol GSH

per 107 erythrocytes, which is also comparable to

previous published reports for human erythrocytes

(Scott et al., 1990).

3.4. Comparison of intracellular GSH

concentrations in distinct feline leukocytes

Next, we used flow cytometry to directly

compare GSH concentrations in different specific

subpopulations of feline leukocytes (Fig. 2). Lym-

phocytes were identified by characteristic forward

and side-scatter pattern and by expression of CD4 or

CD8 for T cells and B220 expression for B cells

prior to determination of intracellular GSH con-

centration. Monocytes were identified by their

characteristic forward and side-scatter pattern, in

addition to cell surface staining for CD14 expres-

sion. Neutrophils were identified by their character-

istic forward and side-scatter pattern, plus

expression of CD14. In Fig. 2A, typical fluorescence

emission histograms are depicted for lymphocytes,

monocytes, and neutrophils. These data illustrate the

pronounced differences in GSH concentrations that

exist between different populations of normal feline

leukocytes. The mean relative GSH concentration

(�S.D.) for each of the major populations of feline

leukocytes was determined by analysis of blood

samples from six healthy cats and is plotted in

Fig. 2B. From these data, it is apparent that feline

neutrophils and monocytes contained significantly

greater concentrations of GSH ( p < 0.001) than

feline lymphocytes. For example, the mean (�S.D.)

GSH MFI for CD4+ and CD8+ lymphocytes was 30

(�6.7) and 20 (�4.4), respectively, while the

mean MFI was 148 (�6.0) for monocytes and

154 (�19.6) for neutrophils. The differences in GSH

concentration between lymphocyte subsets (for

example, CD4+ versus CD8+ T cells) or between

T cells and B cells were not significantly different

( p > 0.05).

A larger series of 26 healthy cats was evaluated to

assess and compare GSH concentrations between

lymphocytes, neutrophils, and monocytes, with the

cell populations defined based on forward and side-

scatter characteristics. In Fig. 3, we found that the

mean GSH content for neutrophils (MFI = 204 � 6.2)

was significantly greater ( p < 0.001) than that for

monocytes (MFI = 155 � 4.3). The mean GSH con-

tent for monocytes and neutrophils was also sig-

nificantly greater ( p < 0.001) than the mean GSH

content of lymphocytes (MFI = 38 � 0.1.4).

3.5. Effects of storage time and temperature on

intracellular GSH concentrations

The effects of storage time and temperature on

GSH content in leukocytes from cat blood samples

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140 135

Fig. 2. Comparison of intracellular GSH concentrations in feline leukocyte subpopulations. Leukocytes were prepared from blood of normal

cats, incubated with mBCl and then analyzed by flow cytometry as described in Section 2. (A) Distinct differences in the mean fluorescence

intensity (MFI; parentheses) for PMN (neutrophils) (top panel) and lymphocytes and monocytes (bottom panel) were revealed after incubation

with mBCl. The y-axis of the histograms represents relative cell numbers. (B) Peripheral blood leukocytes from nine normal cats were first

immunostained with cell surface antibodies, then incubated with mBCl and analyzed by flow cytometry as described in Section 2. The average

MFI (�S.D.) for the five relevant cell population is depicted. * denotes significant differences ( p < 0.001) between CD14+ monocytes and the

three lymphocyte subpopulations (CD4+, CD8+, and B220+ cells), while ** denotes significant differences between neutrophils and the three

lymphocyte populations, as determined by ANOVA and Tukey multiple means comparison. Similar results were obtained in one additional

experiment. (C) GSH concentrations were determined in erythrocytes, lymphocytes, and neutrophils from nine healthy cats by means of mBCl

and flow cytometry. The mean (�S.D.) MFI for each cell population was calculated and plotted. * denotes significant differences ( p < 0.001)

between lymphocytes and neutrophils and ** denotes significant differences ( p < 0.05) between erythrocytes and lymphocytes, as determined

by ANOVA and Tukey multiple means comparison.

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140136

Fig. 3. Relative concentration of GSH in feline neutrophils, mono-

cytes, and lymphocytes. Blood samples from 26 healthy cats were

analyzed using flow cytometry and mBCl to assess differences

between neutrophils, lymphocytes, and monocytes. Each data point

represents a sample from an individual cat. Statistical analysis

(ANOVA) revealed significant differences between monocytes

and neutrophils (*p < 0.01) and between monocytes and lympho-

cytes (**p < 0.001).

Fig. 4. Effects of blood storage conditions on GSH concentrations

in cat leukocytes. Blood samples from eight healthy cats were

collected into EDTA tubes, which were then stored for the indicated

temperature and time. Blood leukocytes were then prepared from

each sample and incubated with mBCl and analyzed by flow

cytometry for quantitation of intracellular GSH concentrations, as

described in Section 2. The average (�S.D.) MFI for each sample

population was plotted. Significant differences in GSH concentra-

tion between any of the four treatment groups were not detected, as

assessed by ANOVA and Tukey multiple means comparison.

were evaluated. Specifically, we assessed the effects

of storage at room temperature for 6 h, storage at

4 8C for 6 h and storage at 4 8C for 24 h on

neutrophil GSH content in EDTA-anticoagulated

whole blood samples obtained from eight healthy

control cats. These experiments demonstrated

that GSH concentrations were stable in EDTA-

blood for up to 24 h, provided the samples were

stored at 4 8C (Fig. 4). In contrast, the GSH content

in neutrophils from blood samples stored at room

temperature for 24 h declined essentially to unde-

tectable levels. For example, the fluorescence

intensity after addition of mBCl to the 24 h samples

was not significantly different from the fluorescence

obtained from untreated control samples (data not

shown).

3.6. Effects of FIV infection on intracellular

leukocyte GSH concentrations

Experiments were done to evaluate the utility of

flow cytometry for detecting changes in leukocyte

GSH content in a feline infectious and immunological

disease model. For these studies, we utilized an FIV

infection model and a pathogenic FIV strain that

rapidly induces immunological disorders in cats

(Avery and Hoover, 2004; Dow et al., 1999; O’Neil

et al., 1996). Nine SPF cats were infected with a

pathogenic clade B isolate of FIV (FIV-2542), which

has previously been associated with rapid disease

onset, monocyte tropism, and maternal virus transmis-

sion (Dow et al., 1999; O’Neil et al., 1996).

Intracellular leukocyte GSH concentrations were

measured 12 weeks after FIV inoculation, at a time

point considered representative of acute infection.

GSH concentrations in leukocytes from FIV-infected

cats were compared to GSH concentrations in

leukocytes (neutrophils, monocytes, and lymphocytes

{CD4+ T cells, CD8+ T cells, and B cells}) from five

age-matched control SPF cats maintained in the same

facility.

We found that there was a significant increase

( p = 0.02) in intracellular GSH concentrations in

neutrophils from FIV-infected cats compared to

control animals (Fig. 5). Monocytes from FIV-infected

cats also had increased GSH concentrations, though

the difference did not reach the level of statistical

significance ( p = 0.06). In contrast, intracellular GSH

concentrations were significantly decreased in CD4+ T

cells ( p = 0.0008) and CD8+ T cells ( p = 0.005) from

FIV-infected cats, compared to age-matched control

animals.

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140 137

Fig. 5. Effects of FIV infection on GSH concentrations in feline lymphocytes, monocytes, and neutrophils. Blood samples were collected from

nine SPF cats infected with FIV and from five age-matched, uninfected control SPF cats and analyzed for GSH concentration 12 weeks after

infection, as described in Section 2. The mean intracellular GSH concentration (MFI 350 nm) for neutrophils, monocytes, CD4+and CD8+ T cells

was calculated and plotted. * denotes statistically differences ( p < 0.05) between cells from control and FIV-infected cats, as assessed by

ANOVA. Similar results were obtained using blood samples obtained from the same cats on one other occasion.

4. Discussion

We have demonstrated in these studies that flow

cytometry combined with the fluorogenic substrate

mBCl could be used to rapidly quantitate intracellular

GSH concentrations in blood leukocytes of cats.

Moreover, we also found that the fluorogenic reaction

elicited by addition of mBCl was specific for

quantitation of GSH within cat leukocytes (Fig. 1).

We also observed that significant differences in

intracellular GSH concentrations exist normally in

the different populations of leukocytes (Figs. 2 and 3).

Intracellular GSH concentrations were stable in

EDTA-preserved blood samples for at least 24 h at

4 8C (Fig. 4), thus increasing the potential clinical

utility of this assay. Finally, we demonstrated that FIV

infection was associated with significant and quite

distinct alterations in intracellular GSH concentrations

in different leukocyte populations from FIV-infected

cats (Fig. 5). Taken together, these results indicate that

flow cytometry can be a rapid and useful means of

assessing intracellular GSH concentrations and by

extension oxidative balance in feline leukocytes.

The tripeptide glutathione (g-glu-cys-gly) is the

predominant low-molecular weight thiol within

mammalian cells, which in the reduced state (GSH)

serves as a major reducing agent for scavenging free

radicals and controlling the cellular redox state (Jones,

2002). In cats, glutathione is particularly important as

an endogenous antioxidant, reducing the intracellular

concentration of reactive oxygen species and con-

jugating with electrophilic metabolic by-products

whose accumulation could otherwise result in cell

membrane lipid peroxidation, enzyme malfunction,

and DNA damage (Fettman et al., 1999). Studies have

shown that glutathione concentrations are signifi-

cantly reduced in the livers of cats with spontaneous

hepatic disease, but we are not aware of other

published reports documenting the intracellular con-

tent of GSH in cats with naturally occurring clinical

diseases (Center et al., 2002).

Therefore, we conducted studies to develop a new

assay for assessing intracellular GSH concentrations.

Use of the flow cytometry-based assay provides a

great deal more information than would otherwise be

obtained using conventional GSH assays, which

typically rely on analysis of lysed samples of whole

blood or tissues. Previous studies of GSH regulation

have focused primarily on the role of erythrocytes,

though recent studies suggest that GSH also plays a

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140138

key role in the regulation of leukocyte function (Droge

and Breitkreutz, 2000). Many of the assays currently

used to determine GSH content in peripheral blood

cannot discern GSH differences between different cell

types, and are often cumbersome, expensive, time

consuming, and sensitive to small changes in assay

conditions (e.g. changes in temperature and pH). In

addition, GSH assays that use blood samples report

their results primarily in terms of erythrocyte GSH

content. Therefore, we assessed and compared the

concentrations of GSH in erythrocytes, lymphocytes,

and neutrophils from cats. Notably, we found that

feline erythrocytes contained significantly less GSH

than feline lymphocytes ( p < 0.05) or neutrophils

( p < 0.001) (Fig. 2C). These results illustrate clearly

that there are normally pronounced differences in

erythrocyte and leukocyte GSH content in cats and

suggest that assessment of total GSH content using

blood samples may be influenced significantly by the

white blood cell count.

The flow cytometric assay is well-suited for the

simultaneous determination of GSH content in

multiple different cell types within a heterogeneous

sample, such as analysis of peripheral blood, body

cavity effusions, bone marrow samples, or cultured

cells (Scott et al., 1990). Monochlorobimane is ideally

suited for use in assays that require live cells, as mBCl

is a non-fluorescent bimane that easily crosses cell

membranes and reacts with sulfhydryl moieties. The

reaction between mBCl and GSH is catalyzed by the

GST enzyme. This reaction with mBCl appears to be

strongly influenced by the specific GST isoenzyme

present in a given species. For example; the use of

mBCl with human leukocytes requires much greater

concentrations, longer incubation times, and results in

greater non-specific binding than in mice, who have

different GST isozymes (Cook et al., 1991). One study

found that in human peripheral blood mononuclear

cells only about one-third of the low molecular weight

fluorescence could be attributed to the mBCl–GSH

adduct, consistent with low GST isoenzyme activity in

human cells (Cook et al., 1991). Under these

conditions in human cells, use of mBCl results in a

high degree of non-specific binding to sulfhydryl

groups on proteins, rather than specific reaction with

free thiol groups such as GSH.

The specificity of mBCl for reactivity with thiol

groups in cats was confirmed through the use of the

specific thiol-depleting agent NEM, which virtually

eliminated all fluorescence from mBCl-treated cat

leukocyte samples (Fig. 1). The major GST isoenzyme

present in cats has not yet been identified, so the effect

of feline GST on catalyzing the interaction of mBCl

with GSH cannot be studied directly in vitro. However,

the rapid increase in fluorescence (20 min) that is

observed in cat leukocytes at very low concentrations

of mBCl is consistent with a GST-catalyzed reaction

with specificity for GSH.

One interesting finding to emerge from our studies

is the large difference in GSH content between feline

lymphocytes and monocytes and neutrophils (Figs. 2

and 3). The marked difference in GSH content

between cat lymphocytes and monocytes and neu-

trophils is similar to the differences in GSH content

noted previously in human leukocytes (Cook et al.,

1991). However, cat neutrophils had the highest GSH

content, whereas human monocytes contain greater

concentrations of GSH than neutrophils (Cook et al.,

1991). Our preliminary studies in dogs indicate that

canine neutrophils and monocytes also contain much

higher intracellular concentrations of GSH than canine

lymphocytes (Webb, CB; unpublished data).

Cats are uniquely susceptible to oxidant injury.

This susceptibility is thought to occur in part because

the cat liver contains relatively low glucuronosyl-

transferase activity, which results in a relative inability

to conjugate oxidative metabolites with glucuronic

acid (Welch et al., 1966). Feline erythrocytes are also

uniquely sensitive to oxidative stress because their

hemoglobin molecule contains 8–10 reactive sulfhy-

dryl groups as opposed to 4 in the dog and other

species. Therefore, the feline hemoglobin molecule is

much more susceptible to disruption following

oxidative insult (Harvey and Kaneko, 1976). Our

studies also indicate that cat erythrocytes contain

relatively little GSH compared to leukocytes. For

example, the GSH content on a per cell basis for

neutrophils is 10 times greater than that of erythro-

cytes (Fig. 2). Therefore, leukocytes may play a more

important role in buffering oxidative stresses in cats

than was previously appreciated.

Decreased concentrations of intracellular GSH have

been found in over 36% of human patients with chronic

diseases including cancer and genitourinary, gastro-

intestinal, cardiovascular, and musculoskeletal diseases

(Lang et al., 2000). HIV infection in humans is

C. Webb et al. / Veterinary Immunology and Immunopathology 112 (2006) 129–140 139

associated with significant changes in intracellular

GSH concentrations in leukocytes, especially in CD4+

T cells (Aukrust and Muller, 1999; Pace and Leaf, 1995;

Staal, 1998). Reduced concentrations of intracellular

GSH may contribute to the pathogenesis of acquired

immunodeficiency syndrome (AIDS). For example,

depletion of GSH in AIDS patients has been associated

with enhanced viral replication in T cells, suppressed

cellular inflammatory response, decreased proliferation

of effector cells, increased lymphocyte apoptosis, and

increased sensitivity to drug toxicities (Pace and Leaf,

1995). Our studies in FIV-infected cats suggest that

infection may also induce significant abnormalities of

glutathione metabolism. Of particular relevance,

intracellular GSH content was significantly decreased

in CD4+ and CD8+ T cells from FIV-infected cats

(Fig. 5). Thus, the FIV-infected cat may provide a useful

model to study the effects of supplementing GSH

precursors (e.g. S-adenosyl methionine) on immune

function in chronically infected animals. It is also

interesting to note that unlike the situation with

lymphocytes, monocyte and neutrophil concentrations

of GSH were actually significantly increased in FIV-

infected cats. In part this response may reflect activation

of innate immune defenses by viral infection. Alter-

natively, these responses may also reflect in part the

unique monocyte tropism of the particular FIV strain

used in these studies (Dow et al., 1999).

In summary, flow cytometric determination of

intracellular GSH concentrations may be a useful new

tool for investigating oxidative balance in cats. The

assay described here can also be performed on

refrigerated blood samples stored for up to 24 h. This

assay may therefore be particularly useful for clinical

or experimental studies assessing the effects of dietary

modifications or supplementation of thiols on oxida-

tive balance and immune function in cats with a

variety of different diseases.

Acknowledgements

The authors wish to acknowledge the technical

assistance provided by Mr. Kevin O’Halloran, Mr.

Mark Mathes, Dr. Kristy Dowers, and Dr. Mike

Lappin. These studies were supported by grants from

the Winn Foundation and from the NIH (R01

AI033773-01).

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