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Klotho−−a Common Link in Physiological
and Rheumatoid Arthritis-Related Aging of
Human CD4+ Lymphocytes
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of June 18, 2017.
Jacek M. Witkowski, Monika Soroczynska-Cybula, Ewa
Bryl, Zaneta Smolenska and Agnieszka Józwik
J Immunol 2007; 178:771-777; ;
doi: 10.4049/jimmunol.178.2.771
http://www.jimmunol.org/content/178/2/771
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References
The Journal of Immunology
Klotho—a Common Link in Physiological and Rheumatoid
Arthritis-Related Aging of Human CD4ⴙ Lymphocytes1
Jacek M. Witkowski,2* Monika Soroczyńska-Cybula,* Ewa Bryl,* Żaneta Smoleńska,†
and Agnieszka Jóźwik*
I
mpaired KLOTHO gene activity is apparently associated with
aging process (1, 2). Klotho knockout mice age faster than
their wild siblings and their phenotype shows multiple features of accelerated aging, including multiorgan failures, greying,
and osteoporosis (1). People exhibiting a polymorphic variant of
KLOTHO gene are more prone to osteoporosis, arterial hypertension, atherosclerosis, emphysema and cognitive impairments, and
might exhibit reduced lifespan (3–7), which directly implicates
Klotho in the mechanisms of human aging, coining it a name of
“aging hormone” (8, 9). It was recently shown that the KLOTHO
gene product possesses ␤-glucuronidase activity (10), although its
precise role in the regulation of intra- and extracellular processes,
and especially participation (if any) of Klotho in the immune cell
function, is still unknown. The “helper” CD4⫹ T lymphocytes of
mammals and man undergo physiological aging, considered a major factor leading to increased loss and/or aberration of the immune
system function with advanced age (11, 12). Characteristic features of the aging peripheral blood CD4⫹ lymphocytes include
reduced proliferative capabilities, changed cytokine secretion patterns and lower proportion of “new emigrants” from the thymus
retaining circular DNA fragments resulting from TCR gene rearrangement (TCR rearrangement excision circles) (13–15). Telomere shortening, reduced TCR repertoire (ability to recognize new
Ags) and accumulation of memory (CD4⫹CD45RO⫹) lympho-
*Department of Pathophysiology, Medical University of Gdańsk, Gdańsk, Poland;
and †Rheumatology Hospital, Sopot, Poland
Received for publication July 14, 2006. Accepted for publication November 7, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Polish State Committee for Scientific Research Grant
P05B 039 22 (to J.M.W.).
2
Address correspondence and reprint requests to Dr. Jacek M. Witkowski, Department of Pathophysiology, Medical University of Gdańsk, Debinki 7, 80-211 Gdansk,
Poland. E-mail address: [email protected]
www.jimmunol.org
cytes and of the CD4⫹ cells that lack CD28, the main costimulatory molecule required for adequate activation of these lymphocytes upon Ag challenge (CD4⫹CD28⫺), with profound changes
in the signal transduction mechanisms, are the cellular background
for the above (14, 16 –20). One of the immune system aberrations
associated with advanced age is an increasing frequency of autoimmune diseases, including rheumatoid arthritis (RA)3—a
chronic, inflammatory disease with very high impact on the quality
of life of a huge proportion (⬃1%) of world population, permanently crippling ⬃30% of sufferers (21). Estimated lifespan of RA
patients is 10 –15 years shorter than that of healthy age-matched
cohort (21). The disease manifests itself as proliferative inflammation of the intraarticular synovial tissue and at its advanced
stages is usually involving progressive destruction of multiple
symmetric joints and adjacent bones. Systemic changes including
aggravated osteoporosis, pleuritis and pericarditis, inflammation of
heart and blood vessels, decreased hematopoiesis, and amyloidosis, may accompany joint destruction and related symptoms (22).
Immunological etiology of RA comprises mainly of impaired
function of the CD4⫹ cells, thought to affect function of synovium
inducing its inflammation (23, 24). Changes are observed both in
the peripheral blood T cells of RA patients as well as in their
counterparts residing in the inflamed synovium; in fact, CD4⫹
lymphocytes were shown to traffic between the blood, synovium
and lymph node compartments (25). Phenotypic changes observed
in the peripheral blood CD4⫹ cell compartment of RA patients
include accumulation of both memory CD4⫹CD45RO⫹ lymphocytes and of the CD4⫹ cells lacking CD28 (CD4⫹CD28⫺) (23,
26). Average number of CD28 molecules per cell is significantly
decreased also in those CD4⫹ cells of RA patients that did not
3
Abbreviations used in this paper: RA, rheumatoid arthritis; CQ, chloroquine; DAS,
disease activity score; DSL, di-saccharolactone; KAAL, Klotho-associated ␣-like sequence; MFI, mean fluorescence intensity.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
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Human CD4ⴙ T lymphocytes undergo aging-related changes leading to decreased immunity to infections and neoplasms, and to
increased frequency of autoimmune diseases including rheumatoid arthritis (RA). Certain changes, observed in the CD4ⴙ cells of
RA patients, resemble those observed during physiological aging, but occur at earlier age. Underlying cellular mechanism(s) of
these similarities are so far largely unknown. Here we show that KLOTHO, a ␤-glucuronidase gene whose activity changes are
associated with aging phenotype, is down-regulated at the mRNA, protein, and enzymatic (␤-glucuronidase) activity levels both
in the healthy elderly and especially in RA CD4ⴙ lymphocytes. Although the exact role of Klotho activity for CD4ⴙ cell function
is unknown, we propose here that it might be involved in anti-inflammatory processes occurring in the young and healthy
individuals, but reduced in both healthy elderly and RA patients. To support this hypothesis, we show here that the reduction of
Klotho expression and activity in both elderly and patients’ lymphocytes occurs in concert with the down-regulation of T cell
costimulatory molecule CD28, the latter known to be dependent on increased levels of TNF-␣. Thus, a common mechanism of
KLOTHO down-regulation, but executed at various times in life, may underlie both physiological and disease-related T cell aging.
Klotho activity might become a target of anti-RA drug development as well as a tool to help increase the immune system efficiency
in the elderly. The Journal of Immunology, 2007, 178: 771–777.
772
loose it completely, due to increased amount of proinflammatory
cytokine TNF shown to reduce the expression of the CD28 gene
(27). Peripheral blood CD4⫹ cells of RA patients exhibit decreased proliferation in response to in vitro stimulation (26, 28),
which is associated with their significantly shorter telomeres (29).
They also contain lower proportion of cells retaining TCR rearrangement excision circles than those of age-matched healthy people (29) and the TCR repertoire is reduced in this cohort (28, 30).
Taken together, the abovementioned features of the peripheral
blood CD4⫹ lymphocytes of RA patients closely resemble those
described during physiological aging, a fact which lead to a suggestion that RA might be related to or even a cause of accelerated
aging of the T cell population (26, 28, 30). In this work we attempt
to investigate the participation of KLOTHO—a gene deeply involved in human aging, but until now not associated with the immune system aging—in physiological and disease (RA)-related aging of human CD4⫹ lymphocytes.
Materials and Methods
Altogether, 14 RA patients aged 21–98 years (average 41.5 ⫾ 19.8 years)
and 15 age- and sex-matched healthy people (average age 41.4 ⫾ 20.4,
range 29 –76 years) took part in the study. Healthy people above the age of
65 years conformed to the SENIEUR Protocol criteria (31). All participants
were informed about the purpose of the tests and gave their written informed consent; the project was approved by the Local Committee for
Biomedical Research Ethics at the Medical University of Gdansk. Diagnosis of RA was based on American College of Rheumatology criteria
(32). Average RA disease activity score DAS28 (32) was 4.5 ⫾ 1.3 (range
2.4 – 6.6; 14.3% exhibited low, 64.3%—moderate and 21.4%— high level
of disease activity at the time of study), while average period of disease
duration since diagnosis was 5.3 ⫾ 5.9 years (range 0 –20 years). Half (7
of 14) of the RA patients were tested at diagnosis and received no specific
treatment at the time of study; another half received specific treatment
consisting of typical doses of corticosteroid alone (2 of 14) or in combination with methotrexate (3 of 14), methotrexate alone (1 of 14) or arechine
(1 of 14) for variable times before the study. Both the patients and healthy
individuals reported sporadic usage of NSAID medication within a month
preceding the study; the frequencies of their use were similar for both groups.
Peripheral blood mononuclear cell isolation, immunomagnetic
purification of CD4⫹ lymphocytes, flow cytometry analysis, and
quantification of CD28 on peripheral blood CD4⫹ cells
For estimation of relative level of expression of CD28 on CD4⫹ lymphocytes, PBMC isolated by flotation over Histopaque (Sigma-Aldrich) cushion were stained with PerCy5-anti-human CD4 (DakoCytomation) and
RPE-anti-human CD28 (BD Biosciences) mAbs at 1 ␮g of Ab per 105 cells
for 30 min at room temperature in the dark, washed with PBS and analyzed
by flow cytometry (FACScan; BD Biosciences). CD4⫹ lymphocytes were
immunomagnetically purified using the Dynal CD4 Negative Isolation Kit
(Invitrogen Life Technologies) according to the manufacturer’s instructions; samples of such purified cells were stained with PerCy5-anti-human
CD4 plus RPE-anti-human CD8 (both DakoCytomation) mAbs for estimation of purity, using analogous staining conditions. Raw data were acquired with dedicated CellQuest software (BD Biosciences) and analyzed
with WinMDI 2.9 (J. Trotter; The Scripps Institute). The mean fluorescence intensity (MFI) of cell-bound PE-anti CD28 Ab was obtained upon
appropriate gating of the CD4⫹ population within the PBMC and adopted
as a measure of CD28 expression.
RT-PCR estimation of KLOTHO gene expression
Total RNA was isolated from 2 ⫻ 106 immunomagnetically purified CD4⫹
lymphocytes per sample using TriReagent (Sigma-Aldrich) and manufacturer’s protocol, and immediately converted to cDNA, using the Improm-II
Reverse Transcription System (Promega). PCRs to detect the products of
KLOTHO and ␤-actin genes were performed using the PCR Core System
(Promega) reagents and thermostable Taq polymerase. Following pairs of
primers were used for KLOTHO: sense 5⬘-GCTTTCCTGGATTGACCT
TG-3⬘, antisense 5⬘-TGTAACCTCTGTGCCACTCG-3⬘; and for ␤-actin:
sense 5⬘-CACCTTCACCGTTCCAGTTT-3⬘, antisense 5⬘-GTCCACCTTC
CAGCAGATGT-3⬘. Amplification of both products was performed using
the Eppendorf Personal Mastercycler and following reaction conditions:
initial denaturation 94°C, 10 min; 30 amplification cycles containing melting for 30 s at 94°C, annealing 30 s at 55°C, and elongation 30 s at 72°C;
after last cycle termination 10 min at 72°C followed by cooling and storage
at 4°C. PCR products were resolved in standard 2% agarose gel with
ethidium bromide and band fluorescence visualized using the GDS-8000
Image Acquisition and Analysis System and LabWorks software (both
from Ultra-Violet Products). Densitometric data obtained for KLOTHO
product bands were standardized using the level of expression of ␤-actin as
housekeeping gene.
Real-time PCR quantification of KLOTHO expression
The same primer pairs as used for RT-PCR were also used for the real-time
PCR. Products of KLOTHO and ␤-actin genes were amplified by PCR as
above to prepare the standard curves for both quantifications. For obtaining enough KLOTHO gene product, total RNA was isolated from
samples of healthy human kidney tissue (33) obtained during elective
surgery. LightCycler and FastStart DNA Master SYBR Green I Kit
(Roche Diagnostics) were used for real time PCR and analysis of raw data
was performed using the LightCycler Software 4.05. The reaction was
performed using 10-min activation at 95°C, followed with 40 cycles composed of 10 s. at 95°C; 5 s at 55°C and 10 s at 72°C each, followed by 30-s
cooling at 40°C. Results for KLOTHO gene product quantitation were presented as proportion of the amount of accumulated gene product to that of
␤-actin gene product.
Western blot analysis of expression of Klotho protein in
isolated CD4⫹ lymphocytes
Protein composition of lysates from two million immunomagnetically purified CD4⫹ lymphocytes per sample was resolved using standard SDSPAGE technique according to Laemmli (34). Proteins were transferred to
nitrocellulose membrane using the Trans-Blot SD SemiDry Transfer Cell
(Bio-Rad), the membrane blocked with 5% no-fat milk and probed for
Klotho using rabbit anti-Klotho affinity pure Ab (Gentaur) at 2 ␮g/ml, or
for actin (gel loading control) using mouse mAb to ␤-actin (Abcam) diluted at 1/1000. Appropriate peroxidase-conjugated anti-Ig Abs and ECL
system (Super Signal West Pico Chemiluminescent Substrate; Pierce) were
used to detect and visualize the proteins of interest. The bands were recorded on x-ray film, digitized using the GDS-8000 instrument and quantified using the LabWorks software (both from Ultra-Violet Products). Relative amounts of Klotho protein were expressed as arbitrary densitometric
units after standardization vs actin content.
Estimation of ␤-glucuronidase activity associated with
Klotho product
Flow cytometric assay to detect and compare Klotho-associated ␤-glucuronidase activity was performed according to Refs. 10 and 35. Briefly, 2 ⫻
105 of the immunomagnetically purified CD4⫹ lymphocytes suspended in
biotin- and flavin-free medium containing 11 mM glucose and 4% FCS in
PBS were incubated with 2 mM fluorescein-di-␤-D-glucuronide for 60 min
at 37°C. The reaction was stopped by fourfold dilution in ice-cold staining
medium, then the cells were immediately washed in ice-cold substrate-free
staining medium and stored on ice until FACS analysis. In separate samples, Klotho-associated ␤-glucuronidase activity was inhibited by preincubation of cells with 5 mM 1,4-di-saccharolactone (DSL), and total ␤-glucuronidase activity by treatment of the cells with 0.3 mM chloroquine (CQ)
(35) for 30 min before addition of fluorogenic substrate and throughout the
reaction time. MFI of cells containing converted substrate was measured
after exclusion of dead cells by propidium iodide staining and subtraction
of background fluorescence of substrate-free cells. The DSL-sensitive
(Klotho-associated) activity of ␤-glucuronidase was expressed as arbitrary
units, calculated as a difference between the background-adjusted MFI corresponding to total (CQ-inhibited) ␤-glucuronidase activity and that obtained for cells pretreated with DSL.
Database search for the sequence homologous to ␣ site of
the minimal promoter of CD28 gene in the vicinity
of KLOTHO gene
Human KLOTHO gene is contained within the 13th chromosome (GenBank
accession no. Z92540). The target ␣ sequence of CD28 minimal promoter (5⬘-ACGTTATATCCTGTGTGAAAT-3⬘) was taken from (27,
36, 37) and inserted in the National Center for Biotechnology Information
BLAST-n online service (38) which was used for online GenBank database
search and comparison of the sequences. A 66% homologous sequence
5⬘-GCTCTATATCCTGTGTCCACA-3⬘ (Klotho-associated ␣-like, KAAL)
was found at 46 kilobase pairs 5⬘ from the initial ATG sequence of the
KLOTHO gene.
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Patients
KLOTHO, ARTHRITIS, AND CD4⫹ LYMPHOCYTE AGING
The Journal of Immunology
773
EMSA comparison of T cell nuclear protein binding to the
␣ and ␣-homologous Klotho (KAAL) sequences
The procedure was performed according to Refs. 36 and 39. The nucleotide
sequences identical to the ␣ and to KAAL were synthesized, 32P-tagged
and applied for EMSA test, using T cell nuclear proteins isolated according
to Refs. 36 and 39. For competition experiments, “cold” nonradioactive
oligonucleotides identical with the ␣, KAAL and irrelevant, nonhomologous sequence corresponding to the Ig switch region recognized by a B cell
specific transcription factor LR1 (40, 41) (5⬘-CCTGGGTCAAGGCTGAA
TAGA-3⬘) were synthesized. These were incubated with the nuclear proteins at concentrations 20, 100, and 300 times exceeding these of radioactive probes, for 1 h before the exposure to 32P probes, according to Refs.
36 and 39.
Statistical analysis
Results
Reduction of KLOTHO gene expression in CD4⫹ cells of
elderly and RA patients
When assessed by RT-PCR, KLOTHO gene is transcriptionally
active in the purified resting CD4⫹ lymphocytes of healthy individuals, and apparently inactive in the same cell type of RA patients (Fig. 1A). It has to be stressed that the amount of the
KLOTHO gene product is rather small even in the cells of healthy
people (⬃1/10th of the product of simultaneously assessed reference gene—␤-actin). Yet even triplication of the amount of cDNA
isolated from the RA CD4⫹ cells in the PCR mixture did not result
in the appearance of visible KLOTHO gene product bands (data
not shown).
To check if apparent lack of the KLOTHO gene product in the
lymphocytes of RA patients was genuine (i.e., the gene was turned
FIGURE 1. Transcriptional activity of KLOTHO gene is significantly
reduced in the CD4⫹ cells of RA patients. A, By RT-PCR, strong bands of
reaction product can be seen in healthy (upper panel) but not in RA material (lower panel). Results for RT-PCR for ␤-actin given as sample uniformity control. Representative results from 4 of 12 pairs (1 to 4, healthy
and RA each) of subjects tested pairwise in separate experiments. B, By
real-time PCR, KLOTHO transcriptional activity relative to that of ␤-actin
gene is significantly reduced in RA material (bars: median ⫾ 25th and 75th
percentile; ⴱ, p ⫽ 0.021 by Mann-Whitney U test, n ⫽ 14 for RA and 15
for healthy group).
FIGURE 2. KLOTHO expression in the CD4⫹ lymphocytes of healthy
individuals inversely correlates with age. Relative KLOTHO expression in
the CD4⫹ lymphocytes assessed by real-time PCR is significantly, inversely correlated with age for healthy (black symbols, r ⫽ ⫺0.51, ⴱ, p ⫽
0.025) but not for RA subjects (open symbols, r ⫽ 0.05, p ⫽ 0.87).
off), or the level of its transcription was below the sensitivity
threshold of the RT-PCR method, we used the real time PCR to
quantify the KLOTHO gene activities in the CD4⫹ lymphocytes
isolated from both healthy and RA individuals. We have found that
by real time PCR the amount of Klotho mRNA standardized to that
of ␤-actin mRNA is extremely low even in CD4⫹ cells of healthy
subjects; however, some transcriptional activity of the KLOTHO
gene in the CD4⫹ lymphocytes of RA patients could in fact be
demonstrated. It was significantly lower than that recorded for the
cells of healthy people (Fig. 1B). We have observed a huge variation in the amount of real time PCR product among the healthy
individuals and, when analyzed as a function of age, a significant
negative correlation could be demonstrated (Fig. 2). No such correlation could be shown between the age and amount of KLOTHO
gene product for RA patients, where even for young subjects the
amount of product was very low (Fig. 2). Although the RA patients
differed with respect to the disease activity (DAS28 score) and
their time from initial diagnosis varied significantly, neither disease activity nor its duration correlated with the levels of KLOTHO
expression assessed by either RT-PCR or real time PCR. Similarly,
FIGURE 3. Klotho protein amount in the CD4⫹ lymphocytes of RA
patients is significantly reduced compared with healthy individuals. A,
Representative (of three experiments yielding similar results) Western blot
detection of Klotho and actin (lane loading control) proteins in CD4⫹ cell
samples from a healthy and two RA individuals. B, Statistical analysis of
the results illustrated in A. The amount of Klotho protein per 1 million
CD4⫹ cells is given as arbitrary densitometric units corrected for actin
contents. Note significant decrease of Klotho protein expression in the cells
of RA patients compared with those of healthy ones (bars: mean ⫹ SD;
p ⫽ 0.033 by two-tailed Student t test for independent data, n ⫽ 3 for each
group). Results for kidney Klotho expression serve as positive control.
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Statistical description, regression analysis and calculations of significance of
observed differences were performed with the help of StatSoft. STATISTICA
(data analysis software system), version 7.1 (www.statsoft.com). Depending
on the normality (or lack of it) of data distribution, averaged values were
presented either as means ⫾ SD or as medians ⫾ 25th and 75th percentile, as
indicated in the figure legends. Accordingly, Student t test for independent data or Mann-Whitney U test were used for assessment of significance of differences.
774
KLOTHO, ARTHRITIS, AND CD4⫹ LYMPHOCYTE AGING
treated” status the difference between both groups was numerically
negligible and statistically insignificant, while that between either
group and healthy remained significant (not shown).
Decreased amount of Klotho protein in RA lymphocytes
At the protein level, we have shown by western blotting that the
amount of Klotho protein in the CD4⫹ lymphocytes of healthy
subjects was at least three to five times lower than in the same
quantity of cellular protein from human kidney, known to contain
detectable amounts of Klotho protein (33) (Fig. 3). In the CD4⫹
cells of RA patients amount of Klotho protein is very low and
significantly decreased in comparison to that seen in the cells of
healthy individuals (Fig. 3). No significant correlations of the
amount of Klotho protein with age of donors could be seen. Interestingly, a second band at ⬃58 kDa molecular mass could be
seen in the samples obtained from both kidney and healthy CD4⫹
cells; this band, probably corresponding to the secretory form of
Klotho (42), was absent from the RA samples (Fig. 3A).
there was no observable influence of pharmacological treatment.
RA patients who were tested at diagnosis, before any specific treatment was started, exhibited the same negligibly low levels of the
gene’s expression by RT-PCR as those methotrexate and/or corticosteroid treated. This notion was corroborated by finding that the
data variability was actually less for the RA samples than for
healthy ones (Figs. 1–3). Also, when the results of real-time PCR
were grouped according to “pharmacologically treated” or “un-
FIGURE 5. Both total and
Klotho-associated ␤-glucuronidase
activities are inversely correlated
with subject’s age. ␤-Glucuronidase activities were estimated as in
Materials and Methods; cf. Fig. 4.
Correlation with age is significant
only for healthy subjects (black
marks; asterisks signify r ⫽ 0.634,
p ⫽ 0.03 and r ⫽ 0.636, p ⫽
0.026, respectively, for total (A)
and Klotho-associated activity (B);
numbers of individuals as in Fig.
4) and not for RA individuals
(open symbols in both A and B).
Klotho-associated ␤-glucuronidase activity is reduced in the
CD4⫹ cells of both healthy elderly and RA individuals
It was recently shown that Klotho might exhibit an enzymatic activity of ␤-glucuronidase (10). Therefore, we have compared both
total (CQ-sensitive) and Klotho-dependent (DSL-sensitive) ␤-glucuronidase (10, 35) activities in the purified CD4⫹ lymphocytes of
healthy and RA individuals, using flow cytometry. We have found
that both total and Klotho-dependent activities of ␤-glucuronidase
are significantly decreased in the CD4⫹ lymphocytes of RA patients compared with healthy individuals (Fig. 4). The proportion
of the latter (putative Klotho-associated ␤-glucuronidase activity)
in the total activity of the enzyme in CD4⫹ lymphocytes was also
significantly reduced in the patients’ cells (0.38 ⫾ 0.16 vs 0.66 ⫾
0.13 in the healthy, p ⫽ 0.05, n ⫽ 12 for each group; Student t test
for independent groups). Klotho-associated activity of ␤-glucuronidase was significantly correlated with the amount of KLOTHO
gene mRNA product (r ⫽ 0.596, p ⫽ 0.04) but only for CD4⫹
lymphocytes of healthy subjects; no such correlation could be
demonstrated for RA cells. Interestingly, both the total and Klothoassociated ␤-glucuronidase activities were significantly, negatively correlated with age, but only in the group of healthy individuals; again, no such correlation could be seen for the cells of
RA patients (Fig. 5).
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FIGURE 4. Both total and Klotho-associated ␤-glucuronidase activities
are significantly decreased in RA patients. A, Representative histograms of
flow cytometric analysis of total (CQ-sensitive) and Klotho-associated
(DSL-sensitive) ␤-glucuronidase activities obtained from purified CD4⫹
lymphocytes of a healthy (upper histograms) and RA subject (lower histograms). Test details are given in Materials and Methods. Control, fluorescence intensity of enzyme-converted fluorescein-di-␤-D-glucuronide in
untreated cells; ⫹CQ, fluorescence of cells treated with 0.3 mM CQ; and
⫹DSL, fluorescence of cells treated with 5 mM DSL for 30 min before and
throughout the fluorogenic enzymatic conversion of the substrate. B, Comparison of total (large symbols) and Klotho-associated (small symbols)
␤-glucuronidase activities (expressed as arbitrary fluorescence units, AU)
in individual healthy (black symbols) and RA subjects (open symbols)
shows significant decrease of both activities in the latter (scatterplot plus
averages shown as mean ⫾ SD; ⴱⴱ, p ⫽ 0.00037, ⴱ, p ⫽ 0.00061, twotailed Student t test for independent data; n ⫽ 12 for healthy and 9 for RA).
The Journal of Immunology
Expression levels of Klotho and CD28 are correlated
Next we attempted to assess the possibility of any relation between
the levels of KLOTHO gene expression and Klotho-associated
␤-glucuronidase activity and the level of CD28 expression on
CD4⫹ lymphocytes. The amount of KLOTHO gene product as
assessed by real time PCR strongly correlated with the relative
level of expression of CD28 (measured as MFI of the CD4⫹
CD28⫹ population) for RA patients but only marginally for
healthy individuals (Fig. 6). No correlation could be demonstrated
between the level of Klotho expression and the proportion of
CD4⫹CD28⫺ lymphocytes for either group under study. On the
other hand, neither total nor Klotho-associated activities of ␤-glucuronidase in CD4⫹ cells of healthy and RA subjects had correlated with the levels of CD28 expression or the proportion of
CD4⫹CD28⫺ cells.
KLOTHO and CD28 genes might be coregulated
It has been recently demonstrated, that the decrease in the expression of CD28 on RA lymphocytes is related to the action of TNF
on the initiator/regulatory element (called the ␣ site) in the CD28
gene promoter (27, 37). Using the GenBank database we have
found a DNA sequence homologous to the ␣ site (Klotho-associated ␣-like, KAAL) at 46-kb pairs 5⬘ from the initial ATG sequence of the KLOTHO gene. The sequences share 66.6% overall
homology, with 100% identity of central 12-nucleotide fragments
(5⬘-TATATCCTGTGT-3⬘), identified for the ␣ site to be the most
important protein-binding part that presumably influence the activity of CD28 promoter (27, 36, 37). To check if the KAAL is
capable of binding any T cell nuclear proteins and if ␣ and KAAL
sequences might bind to similar protein repertoire we have performed the EMSA and competitive EMSA experiments. The result
of a representative EMSA experiment is shown in Fig. 7A. It can
be seen that both ␣ and KAAL sequence bind T cell nuclear proteins of apparently the same molecular mass, although KAAL is
also binding protein(s) or their complexes with lower molecular
weights. There is also some degree of competition for the nuclear
proteins between both sequences, as can be assumed from the results of competitive EMSA experiments in which preincubation
FIGURE 7. KAAL oligonucleotide sequence binds approximately the
same mw. proteins from the T cell nuclei as ␣ sequence from minimal
promoter of the CD28 gene and can compete with the latter for these
proteins. The EMSA experiments were performed as described in Materials and Methods. A, A representative EMSA result (one of three yielding
similar results). T cell nuclear proteins were exposed to: KAAL, 32P-tagged
oligonucleotide identical with KAAL; ␣, 32P-tagged oligonucleotide identical with the ␣ sequence found in the minimal promoter of CD28 gene. In
the competitive EMSA experiments, T cell nuclear proteins were exposed to just the 32P-tagged ␣ oligonucleotide (None) or to the same
after preincubation with a given amount (20, 100, and 300 times that of
the radioactive probe) of competing nonradioactive oligonucleotide:
KAAL or irrelevant nonhomologous oligonucleotide LR1 (LR1⬘) (B)
and LR1 or ␣ (C).
with “cold” KAAL oligonucleotide reduces binding of T cell nuclear proteins to the ␣ sequence (Fig. 7B).
Discussion
Growing amount of data is pointing toward the possibility that RA
is associated with (or even stronger, a cause or consequence of)
accelerated, precocious aging of the CD4⫹ T lymphocytes (26,
28). The mechanisms of T cell aging itself, despite extensive effort
of multiple groups, are still largely elusive (reviewed in Refs. 43–
45). Thus, observed parallelisms between the behavior of CD4⫹
lymphocytes in healthy elderly and in the RA patients are mostly
descriptive and not answering to the question of underlying—possibly common— cause(s). Specifically, although the number of
genes possibly involved in the process of aging has been variably
reported as hundreds or even thousands (46), there are only very
few of them for which a direct participation in aging mechanisms
had been demonstrated. One of these few is KLOTHO, known to
participate in the aging-related phenomena in both mice and men
(1–7), whose protein product had been recently named the “aging
hormone” (8, 9). Data on possible participation of KLOTHO in the
immune cell function are so far at most fragmentary and concerning murine immune system only; thus, it was shown that spleen
and other immune organs of KLOTHO knockout kl⫺/kl⫺ mice are
underdeveloped and that B cell development and differentiation is
impaired (1, 47). Currently, there are no published data on Klotho
expression and/or involvement in the function of either murine or
human T lymphocytes. In this work we have demonstrated that
Klotho expression at the mRNA level significantly decreases in
resting human CD4⫹ lymphocytes proportionally to advancing age
and that it is heavily suppressed in the CD4⫹ cells of RA patients
regardless of the patients’ age. We have also shown the reduction
of Klotho protein level and, finally, the decrease of Klotho-associated ␤-glucuronidase activity in the RA patients’ lymphocytes.
Neither the KLOTHO gene transcriptional activity nor the enzymatic activity of Klotho-associated ␤-glucuronidase in the patients’ lymphocytes was influenced by (sometimes prolonged)
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Transcriptional activity of KLOTHO gene is correlated with
CD28 expression. KLOTHO transcriptional activity was measured by realtime PCR and levels of KLOTHO gene product are shown relative to ␤-actin as in Fig. 1. Levels of CD28 expression on CD4⫹ cells are shown as
arbitrary units of MFI of PE-antiCD28 —stained CD4⫹ cells determined by
flow cytometry. Activity of KLOTHO and expression of CD28 are significantly correlated for CD4⫹ cells of RA (open symbols, r ⫽ 0.64, ⴱ, p ⫽
0.018) but not healthy subjects (black symbols, r ⫽ 0.47, p ⫽ 0.14).
775
776
fected by the enzymatic activity of Klotho ␤-glucuronidase remain
mostly unknown. It was shown that the enzyme hydrolyzes and
activates the TRPV5 channel conducting K⫹ (52) and hydrolyzes
steroid ␤-glucuronides (10). Channels belonging to the TRPV family are present in the T cell membranes and might be involved in
the immune cells’ activation (53). Thus, possible lack of their activation related to decreased or even nullified Klotho activity could
be one of the causes of impaired activation of the T cells both in
healthy aged people as well as in RA patients. Also another postulated substrate, the steroid ␤-glucuronides, may be of interest.
Although cited work described only estrogen glucuronides as experimental Klotho substrates, ␤-glucuronides are also among the
main corticosteroid metabolites (54). Klotho activity, removing the
glucuronide residue, was recently suggested to shift the balance
toward increased levels of unconjugated, active forms of steroid
hormones (10). If this action should include cortisol and the kin,
the indirect effect of Klotho would be anti-inflammatory (by tipping the balance toward increased free corticosteroid levels) and
vice versa, decreased activity of Klotho would be associated with
increased proinflammatory potential. In fact, low grade, partially
subclinical inflammation has recently been postulated as a typical
aging-associated feature of the immune system called inflammaging (55). Interestingly, statins, inhibitors of HMG-CoA reductase used to decrease serum cholesterol levels, were recently
shown to have also some anti-inflammatory properties and to be
beneficial in RA (56, 57). This activity might in some way be
associated with Klotho, as it was shown that statins induce Klotho
expression (58).
Acknowledgments
We are indebted to professor Jorg Goronzy at Mayo Clinic (Rochester,
MN). for allowing us to perform the pilot EMSA experiment in his laboratory. We also thank Anna Budzyńska for excellent technical help.
Disclosures
The authors have no financial conflict of interest.
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pharmacological treatment or by the level of activity of the disease
assessed with the DAS-28 score. In our opinion these finding indicate that the phenomenon of reduction of Klotho expression and
associated enzymatic activity is characteristic for the disease itself
and thus possibly (causatively?) involved in its mechanism, rather
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As we have shown, reduced Klotho mRNA levels are correlated
with the level of CD28 expression on the CD4⫹ lymphocytes in
both RA and (to a lesser extent) healthy lymphocytes. Lower level
of correlation seen for healthy individuals might be due to lesser
variability of CD28 expression on CD4⫹ cells in this population,
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We have found a sequence (KAAL) homologous to the ␣ site
controlling the activity of CD28 gene promoter in the vicinity of
the KLOTHO gene and shown by EMSA and competitive EMSA
experiment that the two sequences might share common, as yet
uncharacterized, regulatory proteins. It is worth noting that the
gene regulation (enhancing or silencing) had been reported over
even much higher distances than that found between the KAAL
sequence and the KLOTHO gene promoter, even up to ⬎300 kbp
(reviewed by Cook (49)). Klotho promoter itself seems to belong
to TATA-less, CAAT-less promoters, heavily regulated by their
ability to bind factors like the Sp-1(50). Thus, these observations
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