proteasome participates in the alteration of signal transduction in … · 2017. 1. 6. ·...

Proteasome participates in the alteration of signal transduction in T andB lymphocytes following trauma-hemorrhage

T.S. Anantha Samy, Martin G. Schwacha, Chun-Shiang Chung, William G. Cio¤,Kirby I. Bland, Irshad H. Chaudry *

Center for Surgical Research, Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Middle House II,593 Eddy Street, Providence, RI 02903, USA

Received 9 September 1998; accepted 23 September 1998

Abstract

Proteasomes are essential components of the cellular protein degradation machinery. They are nonlysosomal and theirparticipation is critical for (1) the removal of short lived proteins involved in metabolic regulation and cell proliferation, (2)the control of the activities of regulators involved in gene transcription, such as nuclear factor-UB (NF-UB) and signaltransducer and activator of transcription (STAT1), and (3) processing of antigenic peptides for MHC class I presentation.Trauma-hemorrhage induces profound immunosuppression which is characterized by reduced splenocyte proliferation,interleukin (IL)-2 and interferon (IFN)-Q productive capacity, increased activation of transcription factors NF-UB andSTAT1 in splenic T lymphocytes, reduced macrophage antigen presentation capacity and inordinate release ofproinflammatory cytokines, such as IL-6 and tumor necrosis factor-K. Furthermore, it appears that the activity of severalregulatory proteins involved in immune function is altered by trauma-hemorrhage. Since proteasomes are involved inregulation and removal of regulatory proteins, we hypothesized that trauma-hemorrhage alters proteasomal activity insplenic lymphocytes. The data showed that activities of 26s proteasome from CD3CD4 and CD3CD8 splenic Tlymphocytes were enhanced following trauma-hemorrhage which was associated with increased expression of NF-UB andSTAT1. On the other hand, trauma-hemorrhage attenuated the activity of 26s proteasome from splenic B lymphocytes whichwas restored upon IFN-Q stimulation and correlated with increased expression of NF-UB. These studies indicate a potentialrole for proteasomes in the regulation of signal transduction in splenic T and B lymphocytes following trauma-hemorrhage,and also suggest them as potential therapeutic targets for attenuation of immune suppression associated with this form ofinjury. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: Trauma-hemorrhage; Splenic lymphocyte; JAK1; Signal transducer and activator of transcription; Nuclear factor-UB;Proteasome

1. Introduction

Proteasomes are high-molecular-mass multi-cata-

lytic ATP-dependent regulatory proteases. They arenonlysosomal and are present in the cytoplasm andnucleus as a hetero-oligomeric complex with an ap-parent sedimentation coe¤cient of 26s [1,2]. Proteo-lytic degradation of proteins with proteasomes oc-curs in the presence or absence of ubiquitin [3^5]and is responsible for the selective removal of un-

0925-4439 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 9 2 5 - 4 4 3 9 ( 9 8 ) 0 0 0 8 9 - 1

* Corresponding author. Fax: +1-401-444-3278;E-mail : [email protected]

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necessary proteins, such as abnormal proteins gener-ated in the cells, transcription factors with rapidturnover rates, and key proteins that are closely re-lated to cell cycle progression and metabolic regula-tion [1,2,6]. Proteasomes are also involved in the ac-tivation of transcription factor, nuclear factor UB(NF-UB). The genes regulated by NF-UB includethose involved in immune responses such as hemato-poietic growth factors, chemokines and leukocyte ad-hesion molecules [7]. The most common form of NF-UB is a heterodimer composed of a p50 and a p65(also termed as Rel A) proteins [8,9]. Proteolysismediated by ubiquitinated proteasomes are requiredfor the activation of NF-UB and its release into thenucleus. The p105, precursor of the p50 subunit ofNF-UB, is processed by ubiquitin-mediated protea-some in the presence of ATP. The NF-UB hetero-dimer, p65^p50, is present in the cytoplasm as a ter-nary complex with the inhibitory protein IUB.Phosphorylation of IUB with a speci¢c kinase [10^12] and subsequent proteolysis mediated by protea-some are essential for the release of NF-UB [13] andtranslocation into nucleus for interaction with specif-ic response elements involved in gene activation.Similarly, proteasomal processing has been reportedto be essential for the negative regulation of signaltransducer and activator of transcription (STAT1),another transcription factor which is also involvedin cytokine gene activation [14]. Furthermore, pro-teasomes are also implicated in antigen processingsince the genes coding for two nonessential protea-some subunits are located within the major histo-compatibility complex (MHC) class II region [15^18] and these genes are inducible by interferon(IFN)-Q for augmented incorporation into proteaso-mal catalytic components [19^21]. Thus, proteasomesappear to have three important regulatory functions:(i) the removal of short-lived proteins, (ii) activationand regulation of transcription factors involved ingene activity, and (iii) processing of antigens forMHCI presentation.

A number of studies have shown that followingtrauma-hemorrhagic injury there is profound im-mune depression [22,23]. Furthermore, the immuno-depression following trauma-hemorrhage is linked toderangement of immune cell functions, includingsplenocytes, observed by depressed mitogenic re-sponses, altered release and activity of cytokines,

and decreased antigen presenting capacity by macro-phages. In a recent study [24], we have observed anenhancement of receptor-initiated phosphorylationand activation of transcription factors, STAT1 andNF-UB, following trauma-hemorrhage in murinesplenic T lymphocytes. Activation of these signalswas further augmented by IFN-Q. Proin£ammatorycytokines released by splenocytes following trauma-hemorrhage appeared to mediate the activation ofthese signals in T lymphocytes. Proteasomes, mayhave an exigent role in hemorrhagic shock becauseof their involvement in the regulation of transcrip-tion factors, NF-UB [13] and STAT1 [14] and in anti-gen processing [25]. Increased expressions of NF-UBand STAT1 in splenic T lymphocytes [24] and de-pression in macrophage antigen presentation [26] fol-lowing trauma-hemorrhage prompted us to evaluatethe activity of 26s proteasome in splenic T and Blymphocytes. Although, involvement of proteasomeshas been observed in T-lymphocyte activation andproliferation [27] and in the processing of antigensfor MHCI presentation by B lymphocytes [4,25],there is very little information available concerningproteasomal activity in vivo and regulation of NF-UB and STAT1 expressions in splenic T and B lym-phocytes following trauma-hemorrhagic shock. Thisinformation is essential since T- and B-lymphocytefunctions are compromised following trauma-hemor-rhage, and modulation of proteasomal activity mayrestore their functions. The present study shows aug-mentation in the activity of 26s proteasomes in theT-lymphocyte CD3CD4 and CD3CD8 subsetsfollowing trauma-hemorrhage. Activation of theCD3CD8 subset correlated with activation ofJAK1, STAT1 and NF-UB expressions and, func-tionally, at the level of cytolytic activity. In B lym-phocytes, increased activity of 26s proteasome wasassociated with increased NF-UB expression insham as well as trauma-hemorrhaged mice followingIFN-Q stimulation.

2. Materials and methods

2.1. Materials, antibodies and bu¡ers

All reagents were of analytical grade. The antibod-ies used were: monoclonal antibodies to JAK1 and

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STAT1 (Transduction Laboratories, Lexington,KY); rabbit polyclonal antiNF-UBp65 and alkalinephosphatase-conjugated antirabbit goat antibody(Rockland, Gilbertsville, PA); alkaline phosphataseconjugated goat antibody to mouse IgG2, £uoresceinisothiocyanate (FITC)-conjugated antiCD3, phycoer-ythrin (PE)-conjugated antiCD4, Cy-chrome (Cyc)-conjugated antiCD8 and Cyc-conjugated antiB220(PharMingen, La Jolla, CA). Recombinant mouseIFN-Q was obtained from RpD Systems, Minneap-olis, MN. Na512 CrO4 (speci¢c activity 512 mCi/mg)was purchased from NEN Life Sciences, Boston,MA. Hexokinase, glucose, ATP, Boc-Leu-Arg-Arg-AMC, Suc-Leu-Leu-Val-Tyr-AMC and Cbz-Leu-Leu-Glu-LNA were obtained from Sigma, St Louis,MO. Lactacystin was from Calbiochem, La Jolla,CA and Tyr-Gly-Arg-CH2Cl was custom synthe-sized. YAC-1 Cells (American Type Culture Collec-tion, Rockville, MD) were grown and maintained inour tissue culture laboratory according to the suppli-er's speci¢cation.

2.2. Animals

Inbred C3H/HEN male mice, 6^8 weeks old andweighing 20^25 g, were purchased from Charles Riv-er Laboratories, Wilmington, MA. Animal studieswere conducted in accordance with the guidelinesset by National Institutes of Health and the proto-cols were approved by the Rhode Island HospitalInstitutional Animal Care and Use Committee. Theprocedures for inducing trauma (laparotomy)-hemor-rhage have been described in detail in earlier publi-cations [24,28]. The mice were randomly assigned totwo groups (n = 3^4 per group). In sham-operatedcontrol the mice underwent same anesthetic and sur-gical procedures, but hemorrhage and resuscitationwere not carried out. In experimental animals traumawas induced by a 2-cm ventral midline laparotomy,followed by hemorrhage consisting of blood with-drawal to 30 mmHg arterial pressure which wasmaintained for 90 min. The animals were resuscitatedwith 4U (volume of blood drawn) Ringer's lactatebu¡er. Pools of 3^4 mice were used in each experi-ment, and each experiment was performed a mini-mum of three times.

2.3. Splenocyte preparation and enrichment ofT-lymphocyte subsets and B lymphocytes

The procedure for the preparation of splenocytesis described in an earlier publication [24]. Enrichmentof T cells was accomplished by passing the spleno-cyte suspension through nylon wool column packedto 10 ml in 20-ml syringes. Enriched T-cell prepara-tion which consisted of both CD3CD4 andCD3CD8 subsets was further fractionated byhigh negative selection chromatography using murineT-cell CD4 subset and CD8 subset column kits(RpD Systems) according to the procedure givenby the manufacturer. The panning procedure ofMage [29] using antimouse Ig antibody coated petridishes was followed for the enrichment of Ig-positiveB lymphocytes from the splenocyte preparation.

2.4. Flow cytometric analysis

To determine the percentage of CD3CD4 andCD3CD8 cells in T-lymphocyte subset prepara-tions and B220 cells in the B-lymphocyte prepara-tion, cells were stained with a combination of anti-bodies conjugated to FITC, PE and Cyc for cellsorting and £ow cytometric analysis. In a typical ex-periment, 2U106 cells were incubated with 10 Wg/mlof nonspeci¢c mouse IgG in PBS containing 1% bo-vine serum albumin and 0.1% sodium azide (PAS)for 15 min on ice. The cells were washed by centri-fugation and incubated with 25 Wl of PAS solutioncontaining 1 Wg of monoclonal antibodies againstCD4 (PE-conjugated), CD8 (Cyc-conjugated) andCD3 (FITC-conjugated) for T cells and B220 (Cyc-conjugated) for B cells. The cells were washed withPBS and ¢xed with 1% paraformaldehyde in PBS(pH 7.4) for 30 min. After ¢xation cells were pelletedby centrifugation and resuspended in 1 ml of PBS,and stored in the dark at 4³C until cyto£uorometricanalysis. Isotypic controls consisting of 1 Wg of eitherFITC, PE, Cyc-conjugated rat IgG2 or FITC-conju-gated hamster Ig per 106 cells were incubated in theexperiment for assessment of non-antibody staining.The multicolor £ow cytometric analysis was carriedout in a 70S spectrum laser (Spectrum Products Di-vision, Palo Alto, CA) operated FACSort (Becton

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Dickinson, Palo Alto, CA) instrument. FITC, PEand Cyc conjugates were excited with argon laserat 488 nm and detected at respective emissions at530 þ 15, 575 þ 15 and 670 nm. Emission overlapswere corrected by electronic compensation.

2.5. Proteasome preparation

Proteasomes (26s) were prepared from enrichedCD3CD4 and CD3CD8 T-lymphocyte subsetsand B lymphocytes using the procedure described byReidlinger et al. [30]. Brie£y, 5^10U107 cells werehomogenized in 10 ml of bu¡er A (20 mM Tris^HCl (pH 7.0) containing 5 mM ATP, 1 mM L-mer-captoethanol, 0.1 mM EDTA and 20% glycerol).After removal of cell debris from the homogenateby centrifugation at 10 000Ug for 60 min, the 26sproteasome was sedimented by centrifugation at140 000Ug for 6 h. The pellet was resuspended inbu¡er A and adsorbed to 10 ml of hydroxyl apatite(Biogel) equilibrated in bu¡er A for 1 h at 4³C. Un-bound proteins were recovered by centrifugation,concentrated with Amicon XM50 membranes (Ami-con, Lexington, MA) and layered on six discontinu-ous gradients of sucrose (10^30% sucrose in bu¡er Awithout glycerol), and centrifuged at 115 000Ug for16 h. The fractions containing the protease activitywere pooled, concentrated again on Amicon XM50membrane and applied to a Sepharose-6 gel ¢ltrationcolumn (Pharmacal 16/50 equilibrated with 20 mMTris^HCl (pH 7.5) containing 2 mM ATP, 1 mMmercaptoethanol, 100 mM NaCl and 10% glycerol),and chromatographed with the equilibration bu¡er.Fractions containing the protease activity werepooled, dialyzed against bu¡er A containing 1 mMdithiothreitol instead of mercaptoethanol, concen-trated with the Amicon XM membrane and storedat 320³C. The protein content of the preparationwas determined by £uorometric assay using NanO-range (Molecular Probes, Eugene, OR). The proteaseactivity of the fractions obtained at di¡erent steps ofthe preparative procedure was monitored by £uoro-metric assay using the synthetic substrate, Boc-Leu-Arg-Arg-AMC as described below.

2.6. Enzyme assays

Peptidase activities were assayed with synthetic

substrates dissolved in 50 mM Hepes bu¡er, pH7.5 or 8.0 [31]. Trypsin-like activity was assayedwith Boc-Leu-Arg-Arg-AMC, chymotrypsin-like ac-tivity with Suc-Leu-Leu-Val-Tyr-AMC and peptidyl-glutamyl peptidase activity with Cbz-Leu-Leu-Glu-LNA. The assays were carried out in 250-Wl reactionmixtures in 96-well microtiter plates (Corning, Corn-ing, NY), and £uorescence measurements were madeon a Bio-Tek FL500 Plate reader (Bio-Tek Instru-ments, Winooski, VT) at 395 nm (excitation) and495 nm (emission). Inhibition of the protease activitywas determined by assaying peptidase activity afterpreincubation of the enzyme with the inhibitors for30 min at 25³C. SigmaPlot and SigmaStat softwares(Jandel, San Rafael, CA) were used for nonlinearregression analysis of data. The hyperbolic equation,f = a*x/(b+x) was used for calculating maximal ve-locity, Vmax, and Km of the proteasome.

2.7. JAK1, STAT1 and NF-UB expressions

The procedure of Imbert et al. [32] was followedfor preparation of cell lysate needed for determiningJAK1 and STAT1 expressions and Dignan et al. [33]for the preparation of nuclear extracts for NF-UBanalysis. Since, NF-UB is released from its ternarycomplex with IUB in the cytoplasm and is immedi-ately translocated into the nucleus (especially afterstimulation), its analysis in nuclear extracts will besynonymous with its activation. NF-UB expressionwas determined by two separate procedures: assayfor transcriptional activity by Western blot analysisand for DNA binding by electrophoretic mobilityshift assay. Monoclonal antibodies to JAK1 andSTAT1 and polyclonal rabbit antiNF-UBp65 wereused, respectively for probing JAK1, STAT1 andNF-UB on the immunoblots. The procedures for im-munoprecipitation, electrophoresis, Western blottransfer and probing of bands with antisera are de-scribed in an earlier publication [24]. The images fortypical blots were captured using a MOCHA II im-age analysis work station (Jandel Scienti¢c, San Ra-fael, CA).

2.8. Electrophoretic mobility shift assay

The procedures for NF-UB probe preparation, itslabeling with [Q-32P]ATP, binding reaction and elec-

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trophoresis are described elsewhere [24]. The gelswere analyzed for radioactivity in InstantImager(Packard, Meriden, CT).

2.9. Measurement of cytotoxic T-lymphocyte activity

The ¢rst basic protocol of Wunderlich and Shearer[34] was followed for induction and detection of cy-totoxic T-lymphocyte activity. For sensitization ofe¡ector cells, T-cell subsets (106/ml in RPMI-10 com-plete media) were treated with 0.5 mg/ml mitomycinC and stimulated in the presence or absence of 10 Wg/ml concanavalin A (Con A), and cultured for 5 daysin a humidi¢ed 5% CO2 incubator at 37³C before usein a 51Cr release assay [34]. Target cells were pre-pared by incubation of 5 ml of rapidly dividing

YAC-1 cells (1U106/ml) in a complete RPMI mediacontaining 100 WCi per ml of Na512 CrO4 (NEN LifeSciences) in a humidi¢ed 5% CO2 incubator for 3 hat 37³C. Following incubation the cells were pelleted,washed three times with media and resuspended inmedia at 1U105 cells/ml. The cytotoxicity assay wascarried out in 96-well u-bottom plates (Corning) inwhich each well contained 104 51Cr-labeled cells in100 Wl. Sensitized T cells in complete medium wereadded to give e¡ector to target cell ratios of 10:1,33:1, 100:1, 333:1, 1000:1, 3333:1 and 10000:1 in atotal volume of 200 Wl. After incubation for 5 h at37³C in a humidi¢ed 5% CO2 incubator, cell-freesupernatants were collected by centrifuging at500Ug for 15 min, and 100-Wl aliquots were countedfor radioactivity in a gamma counter (Wallac, Gai-

Fig. 1. Flow cytometric analysis of T-lymphocyte CD3CD4 and CD3CD8 subsets. T-Cell subsets from antiCD4 and antiCD8columns were reacted with FITC-conjugated-antiCD3, PE-conjugated antiCD4 and Cyc-conjugated antiCD8 antibodies. Multicol-or analysis of stained cells was assayed by cyto£uorometry. Histograms show that cells from the antiCD8 column (A and B) orantiCD4 column (C and D) were s 85% CD3CD4 (A and B) or CD3CD8 (C and D), respectively. Sham, A and C; trauma-hemorrhage, B and D. x-Axis, FITC-antiCD3, emission 525 nm; y-axis, PE-antiCD4, emission 575 nm and Cyc-antiCD8, emission670 nm.

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thersburg, MD). All incubations were carried out intriplicate. The results are expressed as the ID50 ofe¡ector cells which was calculated from the percent-age of speci¢c lysis, at di¡erent e¡ector to target cellratios, according to the formula: % speci¢c lysis =(experimental release3spontaneous release)/(max-imal release3spontaneous release). The spontaneousrelease in 3 h was 6 10% of the maximal releaseinduced by 5% Triton X-100.

2.10. Statistical analysis

SigmaStat, version 2.0 (Jandel, San Rafael, CA),was used for descriptive statistics, one-way analysisof variance and complete comparison Student^New-man^Keuls analyses. Data are expressed as themean þ standard deviation. P6 0.05 was consideredsigni¢cant.

3. Results

3.1. Purity of T-lymphocyte subset and B-lymphocytepreparations

The purity of the T-lymphocyte subsets enrichedby chromatography on antiCD4 and antiCD8 col-umns were determined by multi color cyto£uoromet-ric analysis using £uorescent-labeled antibodies forthe respective subsets. The data presented in Fig. 1demonstrate that the T lymphocytes from antiCD8column were s 85% CD3CD4 and from antiCD4

Fig. 3. The activity (A) and properties (B) of 26s proteasomeprepared from enriched splenic T lymphocytes. The assays werecarried out in a 200 Wl reaction mixture pH (7.5), containing 50mM Hepes/KOH, 50 WM Boc-Leu-Arg-Arg-AMC, and 1 Wgproteasome preparation. The proteasome preparation wastreated with the inhibitors (100 WM) for 30 min at 4³C beforeadding to the assay mixture. C, control; H, 5 units/ml of hexo-kinase+5 mM glucose; H+ATP, hexokinase+glucose+2 mMATP; CE, Tyr-Gly-Arg-CH2Cl; LCY, lactacystin. P6 0.05.

Fig. 2. Flow cytometric analysis of B lymphocytes for B220epitope. Cell suspensions obtained after panning splenocytes onmouse antiIgG antiserum were reacted with Cyc-conjugatedB220 antibody and analyzed by cyto£uorometry. The histogram(Cyc-antiB220 £uorescence, emission 670 nm vs. cell size)shows s 90% were B220. Analysis was similar for B220 lym-phocyte preparations from sham and trauma-hemorrhaged ani-mals (data not shown).

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column were s 90% CD3CD8. Cross-contamina-tion of each lymphocyte subset in sham and trauma-hemorrhage preparations was less than 15%.

The percentage of B220 cells in the B-lymphocytepreparation obtained by panning of splenocytes withantimouse IgG-coated plates was greater than 90%(Fig. 2). The cyto£uorometric analysis of B-lympho-cyte preparation, however, indicated heterogeneity insize distribution due to the presence of granular lym-phocytes. There were no CD3 cells in the B-lym-phocyte preparation (data not shown).

3.2. Properties of 26s proteasome prepared fromenriched splenic T lymphocytes

The rate of hydrolysis of Boc-Leu-Arg-Arg-AMCby proteasome prepared from splenic CD3 T lym-phocytes is shown in Fig. 3A. The proteasomepreparation showed characteristic Michaelis^Mentenkinetics over a wide range of concentrations of thesubstrate with a Vmax of 820 (£uorescence) and aKm value of 53 WM. Treatment with hexokinase(5 units/ml) and glucose (5 mM) inhibited s 70%of the peptidase activity (Fig. 3B). Addition of2 mM ATP completely restored the activity. Thecompetitive inhibitor, Tyr-Gly-Arg-CH2Cl (100 WM),and the microbial peptide, lactacystin (100 WM),

abolished s 90% of the peptidase activity. Thepreparation thus displayed properties of a protea-some and not that of lysosomal peptidase or cyto-plasmic endonuclease.

3.3. Peptidase activities of 26s proteasomes isolatedfrom T-lymphocyte subsets oftrauma-hemorrhaged mice

After ascertaining that the 26s proteasome pre-pared from T lymphocytes exhibited characteristicstypical of a proteasome, properties of proteasomesprepared from the T-cell subsets were compared.The trypsin-like, chymotrypsin-like and peptidyl-glu-tamyl peptidase activities of 26s proteasomes fromthe T-lymphocyte subsets of sham and trauma-he-morrhaged mice are shown in Fig. 4. The proteasomepreparations exhibited kinetics characteristic of reg-ulatory enzymes. Km values were nearly similar forall proteasome preparations irrespective of the T-cellsubset, trauma-hemorrhage insult or the substrate.However, di¡erences were observed in Vmax and thespeci¢c activities (Table 1). CD3CD8 lymphocytesfrom sham and trauma-hemorrhage groups exhibitedslightly higher proteasomal activity when comparedto CD3CD4 lymphocytes in trypsin-like and chy-motrypsin-like activities. Trauma-hemorrhage aug-

Fig. 4. Activities of 26s proteasomes prepared from CD3CD4 and CD3CD8 lymphocytes of sham and trauma-hemorrhage mice.The assays were carried out in a 200 Wl reaction mixture (pH 7.5), containing 50 mM Hepes/KOH, di¡erent concentrations of syn-thetic peptide substrates and 1 Wg proteasome preparation. CD3CD4 : sham (F), trauma-hemorrhage (E) ; CD3CD8 : sham (b),trauma-hemorrhage (a). The error bars represent the standard deviation, n = 3^4.

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mented proteasomal activity in both the subsetsand the CD3CD8 lymphocytes of trauma-hemor-rhage group appeared to posses the highest level ofactivity.

3.4. JAK1, STAT1 and NF-UB expressions inCD3+CD4+ and CD3+CD8+ subsets followingtrauma-hemorrhage

The expression of JAK1, and STAT1 in the cyto-sol and NF-UB in nuclear extracts of CD3CD4

and CD3CD8 cells from sham and trauma-hemor-rhaged mice was determined by Western blot analysis(Fig. 5). Increased expression of all three signals wasobserved in the CD3CD8 cells from trauma-hemorrhaged mice. Conversely, the trauma-hemor-rhage group showed reduced expression of JAK1and STAT1 in CD3CD4 lymphocytes comparedto expression in lymphocytes from the sham group.Increased NF-UB expression, as determined by West-ern blot appeared to be restricted to the CD3CD8

subset of trauma-hemorrhaged mice (Fig. 5). How-ever, analysis by electrophoretic mobility shift assay(Fig. 6) indicated increased NF-UB expression also inCD3CD4 lymphocytes of trauma-hemorrhagedmice.

3.5. Cytotoxic activities of CD3+CD4+ andCD3+CD8+ lymphocytes

Experiments were undertaken to determinewhether trauma-hemorrhage altered the cytotoxic ac-tivity of the T-cell subsets. The cytotoxic response of

Fig. 5. Western blot analysis of cell lysates from CD3CD4

and CD3CD8 lymphocytes of sham (S) and trauma-hemor-rhage (TH) mice for JAK1, STAT1 and NF-UB expressions.Cell lysates were immunoprecipitated with respective antibodiesand adsorbed to protein A Sepharose. The gel pellets, afterwashing, were extracted with Laemmli's sample bu¡er and ali-quots (5 Wg protein) electrophoresed on SDS^PAGE, trans-ferred to nylon membranes and probed with respective antibod-ies. The molecular masses of the protein standards areindicated with an arrow.

Table 1Properties of proteasomes from CD3CD4 and CD3CD8 T-cell subsets of sham and trauma-hemorrhaged mice

Km Vmax v

Trypsin-like activityCD3CD4 Sham 28.5 500 0.56

Trauma-hemorrhage 25.2 780 0.88CD3CD8 Sham 29.5 580 0.68

Trauma-hemorrhage 23.6 810 0.98

Chymotrypsin-like activityCD3CD4 Sham 30.2 380 0.48



Peptidyl-glutamyl peptidase activityCD3CD4 Sham 28.2 290 0.36



The data on Km (WM), Vmax (£uorescence) and speci¢c activity, v (Wmol substrate cleaved per Wg protein per h) were derived from theplots in Fig. 4 by nonlinear regression and rectangular hyperbolic function analysis.

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T-cell subsets from sham and trauma-hemorrhagedanimals, against YAC-1 cells, is shown in Fig. 7. Theresults showed that although low levels of cytotoxicactivity were observed in non stimulated e¡ectorcells, stimulation with Con A increased the cytotoxic

response. An increased cytotoxic response with ConA was observed in CD3CD4 cells of both thesham and trauma-hemorrhage groups. However,Con A stimulation of CD3CD8 cells greatly in-creased the cytotoxic response only in cells fromthe trauma-hemorrhage group, and this increased cy-tolytic activity of CD3CD8 cells was the highestcompared to all the other groups.

Fig. 8. Activities of 26s proteasomes prepared from B220 lymphocytes of sham and trauma-hemorrhage mice. The assays were car-ried out in a 200 Wl reaction mixture (pH 7.5), containing 50 mM Hepes/KOH, di¡erent concentrations of synthetic peptide substratesand 1 Wg proteasome preparation. B220 lymphocytes were stimulated with (+) or without (3) 100 units/ml of IFN-Q for 1 h at 37³C.CD3CD4 : sham (F), sham+IFN-Q (E), trauma-hemorrhage, (b), trauma-hemorrhage+IFN-Q (a). The error bars represent the stand-ard deviation, n = 3^4.

Fig. 7. Cytotoxic activity of T-lymphocyte subsets. 51Cr-LabeledYAC1 cells were used as target cells. CD3CD4 andCD3CD8 cells from sham (S) and trauma+hemorrhage (TH)mice were sensitized with mitomycin C with (+) or without (3)Con A prior to mixing with target cells in the 51Cr release as-say. The e¡ector activity on the y-axis is ID50. The error barsrepresent the standard deviation, n = 3^4. P6 0.05.

Fig. 6. NF-UB expression in nuclear extracts of CD3CD4

and CD3CD8 lymphocytes from sham (S) and trauma-hem-orrhage (TH) mice by electrophoretic mobility shift assay. Ali-quots containing 5 Wg protein were used in each analysis. Thedetails of the assay procedure are given in Section 2. C, con-trol ; A, CD3CD4 cells ; B, CD3CD8 cells.

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3.6. Peptidase activities of 26s proteasomes isolatedfrom B lymphocytes of trauma-hemorrhaged mice

The peptidase activities of 26s proteasome, on thebasis of Vmax and speci¢c activity, v, was lower in Blymphocytes (Fig. 8 and Table 2) as compared to T-cell subsets (Fig. 4 and Table 1). A decrease in thetrypsin-like and chymotrypsin-like activities and anincrease in the peptidyl-glutamyl peptidase activity ofproteasomes was observed in B lymphocytes fromtrauma-hemorrhaged animals compared to sham.IFN-Q treatment of B lymphocytes resulted in an in-crease of trypsin-like and a decrease of peptidyl-glu-tamyl peptidase activities in both sham and trauma-

hemorrhage groups. The chymotrypsin-like activityincreased in the trauma-hemorrhage group and notin the sham group.

3.7. NF-UB expression in B lymphocytes

Expression of NF-UB in nuclear extracts of B lym-phocytes was determined by Western blot (Fig. 9)and electrophoretic mobility shift assay (Fig. 10).The results showed decreased expression of NF-UBin B lymphocytes from trauma-hemorrhaged ani-mals. However, stimulation with IFN-Q increasedthe NF-UB expression in both the sham and trau-ma-hemorrhage groups.

4. Discussion

Intracellular proteolysis serves as an importantcontrol mechanism of regulatory proteins, sincethey are metabolically labile and rapid changes intheir concentration demand a short half-life. Thenumber of proteases involved in this process is not

Fig. 10. NF-UB expression in nuclear extracts of B220 lym-phocytes from sham (S) and trauma-hemorrhage (TH) mice byelectrophoretic mobility shift assay. Aliquots containing 5 Wgprotein were used in the analysis. Stimulation with IFN-Q ( þ )was carried out by treatment of cells with 100 units/ml for 1 hat 37³C.

Fig. 9. Western blot analysis of NF-UB expression in cell lysatesof B220 lymphocytes from sham (S) and trauma-hemorrhage(TH) mice. The details of the immunoblot procedure are givenin Section 2. Aliquots containing 5 Wg (1U) and 10 Wg (2U)protein were analyzed. B220cells were stimulated with (+) orwithout (3) 100 units/ml of IFN-Q (I) for 1 h at 37³C.

Table 2Properties of proteasomes from B220 cells of sham and trau-ma-hemorrhaged mice

Km Vmax v

Trypsin-like activitySham 52.6 235 0.45Trauma-hemorrhage 58.2 207 0.39Sham+IFN-Q 44.6 310 0.52Trauma-hemorrhage+IFN-Q 48.5 371 0.78

Chymotrypsin-like activitySham 28.8 155 0.52Trauma-hemorrhage 24.1 115 0.43Sham+IFN-Q 26.9 150 0.56Trauma-hemorrhage+IFN-Q 22.9 237 0.72

Peptidyl-glutamyl peptidase activitySham 31.2 190 0.50Trauma-hemorrhage 28.0 277 0.57Sham+IFN-Q 33.5 145 0.41Trauma-hemorrhage+IFN-Q 35.8 158 0.43

The data on Km (WM), Vmax (£uorescence) and speci¢c activity,v (Wmol substrate cleaved per Wg protein per h) were derivedfrom the plots in Fig. 8 by nonlinear regression and rectangularhyperbolic function analysis.

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known and is being examined in many investigationsincluding the pathological condition emerging fromtrauma-hemorrhage where in£ammatory stimuli ac-celerate the turnover of many regulatory proteins.Surprisingly, the number of endopeptidases presentin cytoplasm and nucleus are very few compared tolysosomal cathepsins and the secreted proteases.However, a number of endoplasmic peptidases havebeen identi¢ed, and they include calpain [35], metal-loproteinase [36], proline endopeptidase [37], severalcaspases [38] and the proteasome. The proteasomecleaves small and large peptides at neutral or slightlybasic pH at the carboxyl side of acidic, basic andhydrophobic residues. Recent studies have shownthat 20s and 26s proteasomes are involved in theregulation of activities of many short-lived proteinsinvolved in signal transduction and in cell division[5,6]. Participation of proteasomes in the activationof transcription factors, NF-UB [13] and STAT1 [14],and in the processing of antigens for MHCI I [25]presentation has been unequivocally demonstrated.

Hemorrhage, which is normally associated withtrauma, causes a pronounced depression in immunefunction subsequent to increased risk of infectionand multiple organ failure [39]. The immunodepres-sion is induced by severe aberrations in immune cellactivity as evidenced by suppressed T-lymphocyteactivity (reduced interleukin (IL)-2 and IFN-Q re-lease), increased release of proin£ammatory cyto-kines IL-6, IL-1L and tumor necrosis factor (TNF)-K by Kup¡er cells, and reduced antigen presentationby macrophages [22,23,28]. Motivating factors ap-pear to be related to perturbations in the signaltransduction mechanisms, such as [Ca2]i immobili-zation [23,26] and enhancement in the expression oftranscription factors, JAK1, STAT1 and NF-UB [24].Proteasomes activate NF-UB by proteolysis of IUB[13], regulate STAT1 activity negatively by proteoly-sis [14], generate MHCI antigenic peptides by limitedproteolysis [25] or rapidly degrade c-fos and c-jun,which have very short half-lives, by proteolysis [40].Based on these observations, proteasome participa-tion both in the activation, regulation and scaveng-ing of regulatory proteins in T and B lymphocytes inresponse to trauma-hemorrhage is anticipated. Im-munosuppression results in activation of several reg-ulatory proteins involved in cell proliferation anddi¡erentiation and their rapid removal is essential

for the maintenance of normal growth and metabo-lism. Concomitantly, many in£ammatory mediatorsare activated by phosphorylation and are degradedrapidly by proteasomes, as in the case of the criticalinhibitory factor IUB for activation of NF-UB [13].Recently, Wang et al. [27] have also reported protea-somes playing a crucial role in T-cell activation andproliferation through multiple mechanisms. Our ear-lier study [24] has shown increased expressions ofcytokine-receptor mediated kinase, JAK1 and tran-scription factors STAT1 and NF-UB in splenic Tlymphocytes following hemorrhage with or withoutlaparotomy. In this study, we observe that the signalactivations are preferential to the CD3CD8 lym-phocyte subset. Furthermore, the increase in signalexpressions is accompanied by augmented activitiesof 26s proteasome and increased cytotoxicity of theCD3CD8 subset of trauma-hemorrhaged animals.Similarly, a link between activities of 26s proteasomeand activation of p65^p50 heterodimer of NF-UBwas evidenced in B lymphocytes. Although p50-c-Rel is the constitutive heterodimer of NF-UB in Blymphocytes, p65^p50 heterodimer is also expressedin the cytoplasm and not in the nucleus [8,41]. Cyto-kines including IFN-Q can stimulate p65^p50 hetero-dimer expression in B cells, which is consistent withour observation. This NF-UB heterodimer expressionwas attenuated in B lymphocytes following trauma-hemorrhage and the expression was restored uponIFN-Q stimulation. The reason for the decrease inNF-UB expression in B lymphocytes of trauma-hem-orrhaged mice is not clear, although the contribu-tion of elevated circulatory levels of TNF-K observedfollowing trauma-hemorrhage for the decreased ex-pression cannot be ruled out [7,9]. On the otherhand, the IFN-Q stimulation of B lymphocytes fromsham mice resulted in augmented NF-UB expression.These changes in NF-UB expressions parallel thechanges in the potentiating of 26s proteasome activ-ity. Involvement of IFN-Q in the regulation of pepti-dase speci¢city of proteasomes is well known [41^43].In the absence of IFN-Q, proteasome possesses onlythe trypsin-like activity which is needed for MHCI Ipeptide loading, whereas upon IFN-Q stimulationproteasome is active in both trypsin-like and chymo-trypsin-like activities essential for the destruction ofunwanted metabolic proteins. However, it remainsunknown whether presentation of antigenic peptides

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to MHCI I is also enhanced in B lymphocytes inresponse to IFN-Q.

In summary, the present results indicate the poten-tiating of 26s proteasomal activities in splenic lym-phocytes following trauma-hemorrhage which corre-lated with the activation of signal transduction inCD3CD8 T lymphocytes and IFN-Q-stimulated Blymphocytes, thus suggesting an important role forproteasome in the regulation of lymphocyte func-tions. Proteasomes are multi-catalytic and are in-volved in both the activation and rapid removal ofregulatory proteins. Unlike lysosomal proteases orother cytoplasmic proteases, the proteasomes arepossessed with the property of cleaving peptidebonds at several distinct sites beside the three estab-lished ones [41]. Furthermore, their catalytic func-tions can be modulated by mediators of immunefunction, e.g., IFN-Q [41^43] or protease inhibitors,e.g., lactacystin [44]. These characteristics make pro-teasomes a suitable candidate for therapeutic manip-ulation to restitute immune depression, a major con-sequence of trauma-hemorrhage.

Acknowledgements

We thank Mr. Paul Mon¢ls of Central ResearchFacilities at the Rhode Island Hospital for help incyto£uorometric analysis and Mrs. Barbara Mon¢lsfor help with the animal experiments. This study wassupported by USPHS Grant GM37127.

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