(Received for publication, June 7, 1994; and in revised form, November 7, 1994)
From the
Although calpain is ubiquitously present in human tissues and is
thought to play a role in demyelination, its activity is very low in
resting normal lymphocytes. To determine the nature of calpain
expression at the mRNA and protein levels in human lymphoid cells, we
studied human T lymphocytic, B lymphocytic, and monocytic lines as well
as peripheral blood mononuclear cells. Stimulation of cells with the
phorbol ester phorbol myristate acetate and the calcium ionophore
A23187 resulted in increased calpain mRNA and protein expression.
Calpain mRNA expression is also increased in human T cells stimulated
with anti-CD3. A dissociation between the increases of RNA and protein
suggested that calpain could be released from the cells; the subsequent
experiments showed its presence in the extracellular environment.
5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible
inhibitor of mRNA synthesis, reduced calpain mRNA levels by
50-67% and protein levels by 72-91%. Its removal resulted
in resumption of both calpain mRNA and protein synthesis.
Cycloheximide, a translational inhibitor, reduced calpain protein
levels by 77-81% and calpain mRNA levels by 96% in activated
THP-1 cells. Interferon- induced calpain mRNA and protein in U-937
and THP-1 cells. Dexamethasone increased mRNA expression in THP-1
cells. Our results indicate that activation of lymphoid cells results
in de novo synthesis and secretion of calpain.
Calpains (EC 3.4.22.17) are neutral proteinases present in all
cells. Their activation requires intracellular calcium levels much
higher than those found in normal resting cells but similar to those
that may be reached by calcium influx accompanying cell activation,
suggesting that calpain expression in resting and activated states may
be different. Among calpain's many substrates are myelin proteins
including myelin basic protein (1) and myelin-associated
glycoprotein (2) . We have reported previously that calpain
isolated from several human lymphoid cell lines degrades myelin basic
protein (3) and may therefore play a role in the demyelination
observed in experimental allergic encephalomyelitis (EAE) ()and multiple sclerosis (MS)(4) . If calpain
participates in lesion development, one of the sources may be the
activated mononuclear cells infiltrating the diseased central nervous
system, a proposal consistent with the occurrence, in the white matter
of animals undergoing EAE, of perivenular monocytic infiltrates prior
to demyelination. A remarkable characteristic of lesion-associated
lymphoid cells in EAE and MS is their high level of activation,
probably resulting from their encounter with myelin
antigens(5) . Activated T cells may participate directly by
secreting calpain and/or indirectly by producing interferon-
(IFN-
), which activates monocytes and stimulates their proteolytic
activity, explaining why IFN-
can induce relapses in MS patients (6) . However, there are no data available on quantitative
studies of calpain expression in lymphoid cells. We have shown
previously that calpain is present in extremely low levels in
nonactivated lymphoid cells and that large numbers of cells are
required to purify this enzyme in vitro(3) . The
objective of this work was to study calpain expression at the RNA and
protein levels during activation of lymphoid cells by (a)
phorbol esters and calcium ionophores, (b) anti-CD3 antibody,
and (c) IFN-
, a physiological activator of monocytes. Our
results show that calpain expression increases upon lymphoid cell
stimulation and that stimulated lymphoid cells release calpain into the
extracellular environment. A preliminary report of this work has been
presented(7) .
Strikingly, the increases in calpain mRNA and protein levels were unparallel. In the monocytic line U-937, induction of calpain mRNA was greater than that of intracellular protein for all durations of cell stimulation with PMA+A23187. Similarly, in the lymphocytic lines, CCRF-CEM, MOLT-3, M.R., and D.S., PMA+A23187 costimulation resulted in a greater increase in calpain mRNA when compared with that in intracellular protein within 2 h. The increase in the amount of calpain mRNA was at least 2-fold greater than that of intracellular calpain protein, raising a possibility that a significant amount of newly produced calpain may be released in the extracellular medium.
Figure 1:
Effect of PMA and A23187 on calpain
mRNA and protein expression in lymphoid cells in serum-free medium.
Cells were treated with PMA (10 ng/ml) and A23187 (5 µM)
for 0.5-4 h in RPMI 1640 medium devoid of fetal calf serum.
Extracellular medium was recovered by centrifugation of the cell pellet
and was studied for the presence of calpain protein (). Cell
lysates were examined for calpain mRNA (
) and intracellular
protein (
) expression. The sum of calpain protein contents in
cell lysates and extracellular media was taken as total calpain protein
content (
).
Figure 2:
Effect of DRB on calpain expression in
lymphoid cells in serum-free medium. Cells were activated with (PMA, 10
ng/ml + A23187, 5 µM) (-
)
for 0.5-4 h in RPMI 1640 medium devoid of fetal calf serum.
Control cells were left untreated (
-
). Cells
were treated with DRB (16 µM) alone (
- - - -
-
) or in combination with (PMA+A23187)
(
-
). Cells were preactivated with
(PMA+A23187) for 1 h, DRB was added 1 h later and left with the
cells up to 4 h (
-
) or was removed at 2 h, and
cells were restimulated with (PMA+A23187) up to 4 h
(
-
). Extracellular medium was recovered by
centrifugation of the cell pellet and was studied for the presence of
calpain protein. Cell lysates were examined for calpain mRNA and
intracellular protein expression. The sum of calpain protein contents
in cell lysates and extracellular media was taken as total calpain
protein content.
When the cells were first activated with PMA+A23187 for 1 h and then treated with DRB for 1-3 h, DRB reduced the expression of calpain mRNA by 9-64%, intracellular calpain protein by 23-54%, extracellular calpain protein by 11-66%, and total (intracellular + extracellular) calpain protein by 24-58% in comparison with that in PMA+A23187-activated cells that were not treated with DRB. The inhibitory effect of DRB was reversible. After its removal, followed by restimulation with PMA+A23187, calpain mRNA and intracellular calpain protein levels increased again by 2.5-fold and 1.4-fold, respectively. The amount of extracellular calpain protein increased by 4.3-fold and that of total (intracellular + extracellular) calpain protein by 1.6-fold. However, in these cells calpain expression did not reach the peak levels seen in activated cells not treated with DRB.
Figure 3:
Panel A, effect of CHX on calpain
expression in nonactivated THP-1 cells. Cells were treated with CHX (20
µg/ml) alone () or in combination with DRB (16
µM) (
) for 0.5-4 h in RPMI 1640 medium devoid
of fetal calf serum. Control cells were left untreated (
).
Extracellular medium was studied for the presence of calpain protein.
Cell lysates were examined for calpain mRNA and intracellular protein
expression. The sum of calpain protein contents in cell lysates and
extracellular media was taken as total calpain protein content. Panel B, effect of CHX and DRB on calpain expression in
activated THP-1 cells. Cells were activated with (PMA: 10 ng/ml +
A23187: 5 µM) (
) in the presence of CHX (20
µg/ml) (
) or of (CHX: 20 µg/ml + DRB: 16
µM) (
) for 0.5-4 h in RPMI 1640 medium
devoid of fetal calf serum. Control cells were left untreated
(
). Cells were preactivated with (PMA+A23187) for 1 h, CHX
was added at 1 h and left with cells up to 4 h (
) or was removed
at 2 h, and cells were restimulated with (PMA+A23187) up to 4 h
(
). Expression of calpain mRNA and protein was studied as
before.
In PMA+A23187-activated cells, CHX alone reduced calpain mRNA levels by 96% and total (intracellular + extracellular) calpain protein levels by 76% in 4 h (Fig. 3B). CHX, in combination with DRB, reduced calpain mRNA levels in these cells by 96% and total calpain protein levels by 81%. When cells were activated with PMA+A23187 during CHX+DRB treatment, calpain mRNA and total protein expression increased 2-fold in 4 h compared with that in cells that were not activated with PMA+A23187 during CHX+DRB treatment (Fig. 3B). However, in CHX-treated cells, calpain mRNA and total calpain protein were much lower (28- and 4-fold, respectively) than in activated cells not treated with inhibitors. When cells were preactivated with PMA+A23187 for 1 h and then treated with CHX+DRB for 1 h, calpain mRNA expression decreased by 78%, and total calpain protein levels decreased by 50% in comparison with preactivated cells that were not further treated with inhibitors. In a separate set of experiments, cells were preactivated with PMA+A23187 for 1 h and subsequently treated with both inhibitors for 1 h which were removed at that time, and the cells were activated again with PMA+A23187 for 2 h. Removal of the inhibitors and restimulation resulted in an 36% increase in calpain mRNA and 10% increase in total calpain protein.
Figure 4:
Effect of IFN- on calpain expression
in THP-1 and U-937 cells. Cells were treated with IFN-
(125, 250,
500, 1,000 units/ml) for 24, 48, and 72 h in RPMI 1640 medium
supplemented with 10% fetal calf serum. Control cells were left
untreated. Cell lysates were examined for calpain mRNA and
intracellular protein expression. Results are expressed as an X-fold increase in calpain expression in comparison with that
in untreated control cells.
Figure 5:
Effect of dexamethasone (DEX) on
IFN--induced calpain expression in THP-1 and U-937 cells. Cells
were treated with dexamethasone (50 nM) alone or in
combination with IFN-
(125 or 500 units/ml) for 48 h in RPMI 1640
medium supplemented with 10% fetal calf serum. Control cells were left
untreated. Cell lysates were examined for calpain mRNA and
intracellular protein expression. Results are expressed as an X-fold increase in calpain expression in comparison with that
in untreated control cells.
When LME-treated PBMC, which were essentially T lymphocytes, were activated with PMA+A23187 for 24 h, calpain mRNA and intracellular protein levels increased by approximately 2.3-fold, and the calpain protein accounted for 70% of that produced by all PBMC, indicating that T lymphocytes are a major source of calpain in activated lymphoid cells. LME pretreatment reduced the PMA+A23187-induced increase of calpain mRNA by 39% and that of intracellular protein by 28%. In the nonactivated PBMC treated with LME, calpain mRNA expression was 57% lower than LME-untreated cells, and intracellular calpain protein levels were lower by 60%, indicating that monocytes may be an important source of calpain in resting lymphoid cells.
Treatment of PBMC with anti-CD3 (0.5 µg/ml) for 24 h to specifically stimulate T lymphocytes via their antigen receptors and mimic physiologic activation of these cells resulted in a 90% increase (p < 0.05) in the expression of µ-calpain mRNA (Fig. 6).
Figure 6:
Effect of anti-CD3 on µ-calpain
expression in PBMC. PBMC (4 10
/culture) were placed
in quadruplicate cultures in the serum-free QBSF-51 medium at a density
of 5
10
cells/ml and treated with 0.5, 1, 2, 5, or
10 µg/ml anti-CD3 monoclonal antibody (OKT-3) at 37 °C for 24
h. Total cellular RNA was isolated and reverse transcribed using
oligo(dT). PCR amplification was performed over 35 cycles using primers
specific for human µ-calpain and
-actin cDNA sequences in the
presence of tracer amounts of [
P]dCTP. A
PhosphorImager was used for quantitative densitometry of PCR products.
Expression of µ-calpain was normalized to that of
-actin as an
internal control. Results are expressed as mean ± S.D. *p < 0.05, Student's t test.
The expression of calpain in nonactivated but permanently proliferating human lymphoid cell lines is low but detectable(3) . However, calpain expression is likely to change when the cells are activated. In favor of this possibility, Murachi (19) reported that among the lymphoid cell lines in which calpain was detected by Western blot, all of the HTLV-I-infected T cell lines expressed calpain very strongly. HTLV-I-infected T cell lines differ from other T cell lines in that they exhibit the characteristics of activated T cells such as a permanent up-regulation of the IL-2 receptor.
Activation of T cells can be achieved in vitro by phorbol esters and calcium ionophores. PMA, a well known activator of lymphocytes and monocytes, activates protein kinase C and leads to induction of c-fos/c-jun genes. The AP-1 complex, a fos-jun heterodimer, acts as a transcription factor that regulates transcription of target genes. Since the 5` region of the gene for the large subunit of calpain contains an AP-1 binding sequence, phorbol esters could potentially induce calpain expression(20) . Our observation of an increase in both calpain mRNA and cytosolic protein after PMA stimulation confirmed this hypothesis regarding the inducibilty of calpain by this agent.
A protein kinase C-associated pathway of gene expression may also be induced by calcium ionophores via formation of inositol trisphosphate. In agreement with this hypothesis, activation with both PMA and A23187 led to a rapid and time-dependent increase in calpain mRNA and cytosolic protein expression in all the cell lines tested. Some differences between the effects of either activator alone were observed; for example, a greater increase in calpain mRNA was obtained with A23187 than with PMA. Discrepancies of this type are known to occur in lymphocytes(22) , suggesting a preferential use of an inositol trisphosphate- or diacylglycerol-associated pathway of induction. In lymphocytic lines MOLT-3, MOLT-4, and M.R., the increase in both intracellular and extracellular calpain protein was greater than that of calpain mRNA at all time intervals. In contrast, monocytic lines THP-1 and U-937 showed greater increases in calpain mRNA than those in protein, indicating that the mechanism(s) of calpain induction and expression may vary among lymphoid cell types.
The pattern of
calpain expression suggested that the enzyme may not remain entirely
cytosolic. During costimulation with PMA and A23187, intracellular
calpain protein and mRNA increased in parallel for the first 2 h, but
after 4 h of activation a dissociation between the increases in calpain
mRNA and protein levels became apparent. The increase in calpain mRNA
levels exceeded that of intracellular calpain protein, which suggested
that calpain may be released from the cytosol into the extracellular
medium. The release of intracellular calpain was not due to cell death,
since there was no reduction in cell number or viability at the end of
each experiment. Cells were also stimulated in serum-free medium to
exclude the possibility of detection of calpain present in FBS. We not
only detected calpain extracellularly but also observed a
time-dependent increase in its amount after cell activation. Calpain is
known to appear on platelet membranes upon cell activation but is not
``shed'' into the surroundings(23, 24) . In
the lymphoid cells tested, calpain was predominantly cytosolic and not
significantly membrane-associated (data not shown). Since calpain lacks
a signal sequence (18) and it is not an integral membrane
protein with a defined transmembrane domain, secretion rather than
shedding appears to be the likely explanation for extracellular
appearance of calpain. IL-1 does not have a signal sequence but is
secreted(25) . The IL-1
-converting enzyme (26) converts cytosolic IL-1 to its secreted form. Processing
by IL-1
-converting enzyme is mandatory for the transport of mature
IL-1
through cell membrane. Whether a similar processing enzyme
also processes calpain (before its release into the extracellular
environment) remains to be established. Nevertheless, the presence of
extracellular, immunoreactive, and biologically active calpain has been
documented previously in the osteoarthritic synovial fluid of knee
joints (27) and cerebrospinal fluid of MS
patients(28) .
To determine whether calpain induction requires active transcription and/or translation, we studied calpain expression in the presence of DRB, a reversible inhibitor of mRNA synthesis, and/or cycloheximide, an inhibitor of protein synthesis. DRB reduced the PMA+A23187-induced increase in calpain mRNA and protein levels in both nonactivated and activated lymphoid cells. Removal of DRB from the cells resulted in the resumption of calpain expression. CHX alone reduced calpain protein expression by 55% in nonactivated cells and by 76% in activated cells. The inhibitory effects of CHX on calpain expression were greater than those of DRB alone. CHX, in combination with DRB, reduced calpain protein levels by 65-80%. CHX did not lead to superinduction of calpain mRNA such as that seen with IL-2 mRNA(11) . A reduction by 96% in calpain mRNA expression was seen in activated cells treated with CHX alone or with DRB. These results suggest that increased calpain expression in activated lymphoid cells requires de novo synthesis of mRNA and protein.
IFN-, produced by activated T lymphocytes of
T
type, is a prominent physiological activator of
monocytes and macrophages which induces synthesis of major
histocompatibility complex class II antigens that serve in antigen
presentation to CD4
T lymphocytes, and secretion of
neopterin, a diagnostic indicator of immune cell activation in
malignancies, allograft rejection, autoimmune disorders, and infectious
diseases(12) . Immunohistochemical detection of IFN-
in
areas of active demyelination (29) and findings of
exacerbations in IFN-
-treated MS patients (6) suggest a
role for IFN-
in immune demyelination. It was, therefore, of
interest to determine its effect on calpain expression. IFN-
induced calpain expression in THP-1 and U-937 cells by severalfold, but
not as much as PMA and A23187. Dexamethasone usually down-regulates
gene expression, but it enhanced the inducive effect of IFN-
(500
units/ml) on calpain production in THP-1 cells. Dexamethasone, tumor
necrosis factor-
, and bacterial lipopolysaccharide are known to
augment IFN-
-induced neopterin production by PBMC(12) .
Whether antigen-induced activation of T cells via the T cell receptor-CD3 complex affects calpain expression has not been documented. Because the intracellular calcium concentration fluctuates normally at submicromolar levels, µ-calpain is more likely to function in cells under physiological conditions. Anti-CD3 antibodies are mitogenic to peripheral blood T cells because they bind to the T cell receptor(30) . We found that they also induce calpain expression. Furthermore, CD3-mediated T cells activation generates phosphoinositides. Both phosphatidylserine and phosphatidylinositol stimulate calpain activation at a lower calcium concentration(31) . (The order of calpain-activating potency among phosphoinositides is phosphatidylinositol bisphosphate > phosphatidylinositol monophosphate > phosphatidylinositol.) Our observation of up-regulation of µ-calpain by anti-CD3 in unfractionated PBMC shows that T cell receptor-specific T cell activation should also lead to calpain expression and bears great significance to its activation by physiological stimuli in normal and disease states, since calpain activation may be achieved at intracellular calcium concentrations in the presence of phospholipids.
The function of increased calpain expression in activated lymphoid cells remains to be established. Calpain is known to generate protein kinase M, an active form of protein kinase C(32) . Both PMA and A23187 are good inducers of protein kinase C in T lymphocytes, and in view of their ability to up-regulate calpain expression in lymphoid cells, induction of calpain may be an intermediate step to protein kinase C activation. Since T lymphocytes harbor 70% of calpain produced by the PBMC pool, induction of calpain expression may have significant effects on T cell activation in normal and disease states.
In
monocytes and macrophages, calpain has been identified as a processing
enzyme for IL-1, allowing its secretion in a biologically active
form(33) . The calcium ionophores A23187 and ionomycin
dramatically enhance the processing and secretion of murine and human
IL-1
by macrophages. Calpain inhibitors (EGTA, leupeptin, and
anti-calpain mAb) inhibit IL-1 processing in activated macrophages, and
calcium ionophores do not induce IL-1 secretion from non-macrophage
cell lines that synthesize but do not normally secrete
IL-1(33) . PBMC show a lag phage of approximately 18 h before
they secrete active IL-1. After IFN-
stimulation it takes
18-24 h for expression and synthesis of calpain in the monocytic
lines THP-1 and U-937. In resting monocytes, IL-1 remains exclusively
intracellular and is not secreted until the macrophages reach full
activation. The very low levels of intracellular calpain in resting
monocytes should not be sufficient to process the IL-1 precursor to an
active, secretable form requiring de novo synthesis of calpain
to process IL-1.
IFN- is known to augment markedly the
production of IL-1, TNF-
, and IL-6 by monocytes. An initial step
of IFN-
-induced macrophage activation may be crucial, especially
when it leads to immune-mediated tissue injury as in IFN-
-treated
cases of MS(6) . Both IFN-
and tumor necrosis factor-
have been shown to promote an inflammatory response in the central
nervous system of rats leading to meningitis and resulting in
demyelination similar to that observed in EAE(34) . In these
cases, activation may also result in calpain secretion. If secreted
calpain is proteolytically active, it may play a significant role in
the immune demyelination of EAE, MS, and HTLV-1-associated
myelopathies.