©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calpain Expression in Lymphoid Cells
INCREASED mRNA AND PROTEIN LEVELS AFTER CELL ACTIVATION (*)

(Received for publication, June 7, 1994; and in revised form, November 7, 1994)

Rajendra V. Deshpande (1)(§) Jean-Michel Goust (1) (2) Arun K. Chakrabarti (2) Ernest Barbosa (2) Edward L. Hogan (2) Naren L. Banik (1) (2)(¶)

From the  (1)Departments of Microbiology and Immunology and (2)Neurology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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) .


EXPERIMENTAL PROCEDURES

Chemicals and Reagents

All chemicals and reagents were purchased from Sigma unless mentioned otherwise.

Cell Lines

CCRF-CEM, MOLT-3, MOLT-4 (T lymphocytes, human leukemia), THP-1 and U-937 (monocyte-like, human histiocytic lymphoma) were obtained from ATCC. The B lymphocyte lines (M.R., D.S.) were established from normal individuals. Cells were cultured in RPMI 1640 medium supplemented with 2 mML-glutamine (Life Technologies, Inc.), 10% heat-inactivated fetal bovine serum (FBS) (HyClone Laboratories), and gentamycin (Life Technologies, Inc.) (1.6 µg/ml). Cell cultures with at least 98% cell viability (tested with trypan blue) were chosen for experimental use. Loss of cell viability at the end of each experiment also was monitored and was less than 3%.

Treatment of Cells with PMA and/or A23187

Lymphoid cells (1-5 times 10^6/ml) were treated with PMA (10 ng/ml) (8) and/or the calcium ionophore A23187 (5 µM) (9) for 0.5-4 h in RPMI 1640 medium with or without 10% FBS. After completion of treatment, cells were washed twice with Hanks' balanced salt solution and used for calpain expression studies.

Treatment of Cells with 5,6-Dichloro-1-beta-D-ribofuranosyl Benzimidazole (DRB) and/or Cycloheximide (CHX)

At least 10^7 cells were treated with the reversible inhibitor of mRNA synthesis, DRB (16 µM)(10) , alone or in combination with PMA+A23187 in serum-free RPMI 1640 medium for 0.5-4 h. Cells were also treated with CHX (20 µg/ml) (11) alone or in combination with either PMA+A23187 or DRB in serum-free medium. Simultaneously, one aliquot of cell suspension was activated with PMA+A23187. Control cells were left untreated. Some cells were preactivated with PMA+A23187 for 1 h; then DRB was added at 1 h and left with cells up to 4 h or was removed after 2 h, and cells were restimulated with PMA+A23187 up to 4 h. Some cells were preactivated with PMA+A23187 for 1 h; CHX was added with or without DRB at 1 h and left with cells up to 4 h or was removed after 2 h, and cells were restimulated with PMA+A23187 up to 4 h.

Treatment of Cells with IFN- and Dexamethasone

Cells were cultured in RPMI 1640 containing 10% FBS and treated with recombinant human IFN- (125, 250, 500, 1,000 units/ml) (Cellular Products Inc.) for 24, 48, and 72 h. Cells were also treated with dexamethasone (50 nM) for 1 h and were left in culture with or without added IFN- (125 or 500 units/ml) for 48 h(12) .

Treatment of Peripheral Blood Mononuclear Cells (PBMC) with PMA, A23187, Leucyl Methyl Ester (LME), and Anti-CD3

PBMC were isolated by density separation using Lymphoprep (Nyegard). Cells (4 times 10^6/ml) were cultured in RPMI 1640 containing 10% FBS and treated with PMA (10 ng/ml) and/or A23187 (5 mM) at 37 °C for 24 h. An aliquot of cell suspension was treated with LME (5 mM) at 37 °C for 30 min to eliminate monocytes(13, 14) . In a separate experiment, PBMC (4 times 10^6) were cultured in the serum-free QBSF-51 medium at a density of 5 times 10^5 cells/ml in the presence of 0.5, 1, 2, 5, and 10 µg/ml anti-CD3 monoclonal antibody (Orthoclone OKT-3, a generous gift from the Ortho Corporation) at 37 °C for 24 h.

Production of Monoclonal Antibody

An anti-calpain monoclonal antibody (mAb) was produced by immunizing BALB/c mice with a 80-kDa subunit of m-calpain, using procedures described previously (15) . The antibody reacted only with m-calpain and did not react with partially purified µ-calpain from erythrocytes.

Enzyme-linked Immunosorbent Assay for Detection of Calpain

Cells were lysed by ultrasonication in 10 mM phosphate-buffered saline, pH 7.4, containing 0.1% Triton X-100 and incubated at 4 °C for 30 min, with vortexing every 5 min. Cell lysates were centrifuged at 14,000 times g for 15 min and diluted to a 0.05% final concentration of Triton X-100. In other experiments, cell supernatants were obtained after stimulation in serum-free medium. Microtiter wells were coated at 4 °C for 24 h with cell lysates or supernatants (200 µl/well), washed with phosphate-buffered saline, and blocked with 1% bovine serum albumin at 37 °C for 1 h. The mouse anti-calpain monoclonal antibody (1:1,000 dilution) was added to all wells at 4 °C for 24 h. After washing the wells, peroxidase-labeled sheep anti-mouse antibody (1:1,000) (ICN Biochemicals) was added at room temperature for 45 min. The substrate, a 0.03% solution of 2,2`-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) in 100 mM citrate buffer, pH 4, containing 0.003% hydrogen peroxide, was added at room temperature for 30 min, and the color development was read at 405 nm in an enzyme-linked immunosorbent assay multiscan spectrophotometer. The amount of calpain protein in samples was determined by using a standard curve generated by entering log (OD) versus log (calpain concentration) using serial dilutions (20 ng to 10 µg) of calpain standard antigen. In all experiments, the r^2 (correlation coefficient) values of standard curves were 0.95-1.00 and p <0.001 to <0.01. The addition of calpastatin to calpain standards did not change mAb binding, indicating that this mAb was suitable for estimation of calpain protein in cell lysates in which some of the calpain could escape detection if bound to calpastatin.

Construction of Calpain cDNA Probe by Reverse Transcriptase-PCR

Five µg of rabbit brain RNA was reverse transcribed 42 °C for 1 h using 200 units of reverse transcriptase and 50 pmol of the downstream primer (bases 1235-1256 of human m-calpain cDNA sequence)(16, 17, 18) . PCR was carried out using l5 units of Taq DNA polymerase and 50 pmol of the upstream primer (bases 616-637). Thermal cycling was for 35 cycles, 1 min at 94 °C, 2 min at 55 °C, and 3 min at 72 °C. The 650-base pair product was cloned into pBluescript SK+ plasmid (Stratagene) and sequenced.

Slot Blots of Total Cellular RNA

Nytran membrane (0.45-µm pore size) was presoaked in 20 times SSC prior to assembly of the Miniblot II slot blot system (Schleicher and Schuell). RNA samples (15-20 µg) were dissolved in 10 µl of sterile water, 20 µl of 100% formamide, 7 µl of 37% formamide, 20 µl of 20 times SSC, and 10 µg of carrier tRNA, incubated at 68 °C for 5 min, and cooled. Four hundred µl of 10 times SSC was added, and the samples were loaded with gentle suction. Each sample was rinsed twice with 10 times SSC. Membranes were air dried, and the RNA was cross-linked by UV irradiation (120J) in a Stratalinker model 1800 (Stratagene).

Hybridization and Autoradiography

Prehybridization and hybridization were carried out in 50% formamide, 5 times SSC, 0.1% SDS, 0.1% and 5 times Denhardt's solution at 42 °C. Calpain cDNA probe was radiolabeled (1 times 10^5 cpm/µl, 7 times 10^9 cpm/µg) (19, 20) using a random primer DNA labeling kit (Boehringer Mannheim). Hybridized membranes were washed twice at room temperature with 2 times SSC, 0.1% SDS, and 0.1% sodium pyrophosphate, and once in 0.1% SDS, 0.1 times SSC at 55 °C. Autoradiograms were developed after 48 h. Analysis of the area and density of the bands on the autoradiograms was performed using Applescan and Image software programs. One unit of RNA was defined as a 6.45 times 10-cm^2 area of a peak representing the intensity and width of a band on the autoradiogram measured by Image software program.

Reverse Transcriptase-PCR for µ-Calpain mRNA Expression

Anti-CD3-treated PBMC were washed twice with phosphate-buffered saline, and total cellular RNA was isolated. Reverse transcriptase-PCR was carried out using the GeneAmp RNA PCR kit (Perkin Elmer). Total cellular RNA (1 µg) was reverse transcribed at 42 °C for 1 h, using oligo(dT) (2.5 µM), and Moloney murine leukemia virus reverse transcriptase (2.5 units/µl). Three-fourths of the reverse transcrption product was taken for PCR amplification with µ-calpain primers (0.5 µM each) and the remaining with beta-actin primers (0.5 µM each) in a 50-ml reaction mixture containing 2.5 units of AmpliTaq DNA polymerase and 0.2 µM [P]dCTP. The primers used in the PCR reaction were: µ-calpain sense (bases 1514-1527), antisense (bases 2187-2210)(21) ; beta-actin sense (bases 1259-1278), antisense (bases 2350-2373)(22) . PCR amplification was performed using the following program: 95 °C/1 min (1 cycle), 94 °C/1 min, 60 °C/1 min 15 s, 72 °C/1 min 30 s (35 cycles), and 72 °C/7 min (1 cycle). Equal amounts of PCR products were resolved on a 1.3% agarose gel. The PCR products were 697 and 538 base pairs for µ-calpain and beta-actin, respectively. The gel was dried and exposed to the PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and the x-ray film (PDB-1, Kodak). Quantitative densitometry of product bands was performed on the PhosphorImager. Expression of µ-calpain was normalized to that of beta-actin as an internal control.


RESULTS

Effect of PMA and/or A23187 on Intracellular Calpain Protein and mRNA Levels of Cells in Serum-supplemented Medium

Calpain expression was first studied in cells stimulated with PMA and/or A23187 from 0.5 to 4 h in serum-supplemented medium (Table 1). A time-dependent increase in mRNA and intracellular calpain protein was observed in all cell lines. PMA and A23187 induced similar levels of calpain mRNA; however, at 4 h the amount of cytosolic calpain protein induced by A23187 was significantly greater (p = 0.028) than that induced by PMA (Table 2). The highest expression of calpain mRNA was observed by costimulation with PMA+A23187, although it was not significantly higher than that due to either reagent alone after 2 h of stimulation. In contrast, the amount of cytoplasmic calpain protein after costimulation with PMA+A23187 was significantly greater than that due to either reagent alone at all time points. The combined effects of PMA+A23187 were additive, not synergistic. In all cell lines, PMA treatment alone resulted in a 0.5-4-fold increase in calpain mRNA and 0.3-5-fold increase in intracellular calpain protein levels. A23187 treatment alone resulted in a 0.7-6-fold increase in calpain mRNA and 0.3-5.7-fold increase in intracellular calpain protein levels. As in most cell types, maximum stimulation (1-13-fold increase in calpain mRNA and 1.3-6.7-fold in intracellular calpain protein levels) was observed with PMA+A23187 costimulation.





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.

Effect of PMA and A23187 Stimulation on Intracellular and Extracellular Calpain Protein and mRNA Levels of Cells in Serum-free Medium

To test the possibility of whether activated lymphoid cells might secrete calpain in the extracellular environment, cells were stimulated with PMA+A23187 for 0.5-4 h in serum-free culture medium, thereby eliminating reactivity of the mAb with FBS in the enzyme-linked immunosorbent assay. Cell lysates and extracellular media were tested for the presence of calpain protein. The results are shown in Fig. 1. Stimulation of MOLT-3, MOLT-4, M.R., THP-1, and U-937 cells with PMA+A23187 resulted in a time-dependent increase in expression of both calpain mRNA (0.6-18-fold) and intracellular protein (1-18-fold). Calpain (70 ng to 5.54 µg/10^6 cells) was also detected in the extracellular medium of nonactivated cells, and its levels increased severalfold (170 ng to 36 µg/10^6 cells) upon stimulation. The increase in extracellular calpain was also time-dependent. MOLT-3, MOLT-4, and M.R. cells showed greater levels of total (intracellular + extracellular) calpain protein than those of calpain mRNA at all time intervals. In contrast, the monocytic lines THP-1 and U-937, when activated, continued to show higher levels of calpain mRNA than protein, similar to what was observed previously with stimulation of these cells in serum-supplemented media.


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 (bullet). Cell lysates were examined for calpain mRNA (bullet) and intracellular protein (bullet) expression. The sum of calpain protein contents in cell lysates and extracellular media was taken as total calpain protein content (bullet).



Effect of DRB on Calpain Expression

In nonstimulated MOLT-3, MOLT-4, M.R., and U-937 cells, DRB reduced calpain mRNA expression by 60-67% in 4 h, but by only 10% in THP-1 cells (Fig. 2). In the lymphocytic lines (MOLT-3, MOLT-4, M.R.) and the monocytic line (U-937) DRB reduced calpain protein levels by 63-95% in 4 h. Calpain production in THP-1 cells was inhibited minimally (10-21%) by DRB. In stimulated cells, treatment with DRB for 4 h uniformly reduced calpain mRNA levels by 65-93% and protein levels by 55-80% in all cell lines (Fig. 2). The inhibitory effect of DRB on calpain mRNA levels was much greater in nonactivated cells, whereas its effect on total calpain protein levels was much greater in activated cells.


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) (bullet-bullet). Cells were preactivated with (PMA+A23187) for 1 h, DRB was added 1 h later and left with the cells up to 4 h (up triangle-up triangle) or was removed at 2 h, and cells were restimulated with (PMA+A23187) up to 4 h (circle-circle). 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.

Effect of CHX on Calpain Expression

THP-1 cells were treated with CHX in the presence or absence of DRB and/or PMA+A23187 in serum-free media for 0.5-4 h. In nonactivated cells, CHX alone reduced calpain mRNA levels by 40% and total (intracellular + extracellular) calpain protein levels by 55% in 4 h (Fig. 3A). This decrease was greater than that observed with DRB treatment alone. CHX, in combination with DRB, reduced calpain mRNA levels by 50% and total calpain protein levels by 65%. When cells were activated with PMA+A23187 during CHX treatment, calpain mRNA expression increased 2.3-fold, and total calpain protein expression increased 3-fold in 4 h compared with that in cells that were not activated with PMA+A23187 during CHX treatment (Fig. 3B). However, in CHX-treated cells, calpain mRNA levels were 22-fold lower and total calpain protein levels were 3-fold lower than those in cells activated with PMA+A23187 but not treated with CHX. CHX treatment did not lead to superinduction of calpain mRNA at any time point. When cells were preactivated with PMA+A23187 for 1 h and then treated with CHX for 1 h, calpain mRNA expression decreased by 36%, and total calpain protein levels decreased by 24% at the end of the 2nd h in comparison with preactivated cells that were not treated with CHX. In another set of experiments, cells were preactivated with PMA+A23187 for 1 h; subsequently, CHX was added to cell culture for 1 h, the inhibitors were then removed, and cells were reactivated with PMA+A23187 for 2 h. Removal of CHX and restimulation of cells resulted in an 11.5% increase in calpain mRNA and 22.3% increase in total calpain protein. Although these levels of calpain expression were lower than those in cells that were not treated with CHX, the increased expression after removal of CHX indicated resumption of calpain synthesis.


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) (circle) 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) (bullet) 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 (circle) or was removed at 2 h, and cells were restimulated with (PMA+A23187) up to 4 h (up triangle). 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.

Effect of IFN- and Dexamethasone on Calpain Expression

IFN- is a direct activator of monocytes. It is known to enhance neopterin secretion by PBMC, a diagnostic indicator of monocyte activation in malignancies, allograft rejection, autoimmune disorders, and infectious diseases, and dexamethasone augments this effect of IFN-(12) . To determine whether IFN- and/or dexamethasone affects calpain expression, THP-1 and U-937 cells were treated with IFN- (125-1,000 units/ml) for 24-72 h. Stimulation of cells with IFN- resulted in a time- and dose-dependent increase in calpain mRNA (Fig. 4). The greatest increase in calpain mRNA levels (7-fold for THP-1 and 8-fold for U-937) was observed in cells stimulated with 1,000 units/ml IFN- for 72 h. IFN- also increased intracellular calpain protein levels in THP-1 and U-937 cells. In THP-1 cells, these levels increased 4.1-fold in 48 h with 500 units/ml IFN- and then dropped for all IFN- concentrations. In U-937 cells, these levels increased 11.9-fold in 48 h with 250 units/ml IFN-. For both cell lines, calpain protein levels reached peak levels at 48 h. In THP-1 cells, dexamethasone enhanced the effect of IFN- (500 units/ml) on calpain protein expression by 1.5-fold (Fig. 5). In U-937 cells, however, dexamethasone affected IFN--induced calpain expression only minimally. The amount of secreted calpain could not be estimated in these experiments, since cells were cultured in serum-supplemented medium for IFN- treatment, and the anti-calpain mAb reacted with calpain in FBS.


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.



Effect of Cell Activation on Calpain Expression in PBMC

Stimulation of PBMC with PMA+A23187 for 24 h increased expression of calpain mRNA by 136%, intracellular calpain protein by 79%, and extracellular calpain protein by 21% compared with untreated controls (Table 3). Intracellular calpain represented 94% of total (intracellular + extracellular) calpain protein in nonactivated cells and 96% of that in activated cells. Calpain protein levels were considerably lower than those seen in lymphoid cell lines.



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 times 10^6/culture) were placed in quadruplicate cultures in the serum-free QBSF-51 medium at a density of 5 times 10^5 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 beta-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 beta-actin as an internal control. Results are expressed as mean ± S.D. *p < 0.05, Student's t test.




DISCUSSION

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-1beta does not have a signal sequence but is secreted(25) . The IL-1beta-converting enzyme (26) converts cytosolic IL-1 to its secreted form. Processing by IL-1beta-converting enzyme is mandatory for the transport of mature IL-1beta 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-alpha, 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-1alpha, 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-1alpha 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-alpha, 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-alpha 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.


FOOTNOTES

*
This work was supported in part by NINDS National Institutes of Health Grants NS-11066 and NS-31622, by National Multiple Sclerosis Society Grant RG 2130-A1, by Paralyzed Veterans of America Grant SCRF-1238, and by a student fellowship from the Medical University of South Carolina (to R. V. D.). This work represented a partial fulfillment of the requirements for the Ph.D. degree of R. V. D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: The James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10021.

To whom correspondence should be addressed: Dept. of Neurology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Fax: 803-792-8626.

(^1)
The abbreviations used are: EAE, experimental allergic encephalomyelitis; MS, multiple sclerosis; INF, interferon; FBS, fetal bovine serum; PMA, phorbol myristate acetate; DRB, 5,6-dichloro-1b-D-ribofuranosylbenzimidazole; CHX, cycloheximide; PBMC, peripheral blood mononuclear cells; LME, leucyl methyl ester; µ-calpain, micro-calpain; m-calpain, milli-calpain; mAb, monoclonal antibody; PCR, polymerase chain reaction; IL, interleukin; HTLV, human T cell leukemia/lymphoma virus.


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