Circulating intercellular adhesion molecule-1 (ICAM-1) as an early andsensitive marker for virus-induced T cell activation
J. P. CHRISTENSEN, J. JOHANSEN, 0. MARKER & A. R. THOMSEN Institute of Medical Microbiology andImmunology, University of Copenhagen, Copenhagen, Denmark
(Acceptedfor publication 26 July 1995)
SUMMARY
The effect of systemic virus infection on the level of circulating ICAM-l (cICAM-l) in serum, andthe role of virus-activated T cells in this context, were studied using the murine lymphocyticchoriomeningitis virus infection as primary model system. A marked virus-induced elevation incICAM-1 in serum was revealed, the presence ofwhich coincided with the phase of virus-induced Tcell activation. However, high levels of cICAM-1 in serum were observed well before maximal Tcell activation could be demonstrated. No increase in cICAM-1 was observed in the serum ofinfected T cell-deficient nude mice, clearly demonstrating that T cells were mandatory. Analysis ofMHC class I and MHC class II-deficient mice revealed that either CD4+ or CD8 + T cells aloneare sufficient, despite a markedly reduced inflammatory exudate in the former animals. Theseresults indicate that virus-activated T cells induce shedding of ICAM-1 into the circulation, andthis parameter may be used as an early and sensitive marker for immune activation.
Keywords intercellular adhesion molecule-h circulating ICAM-1 in serum LCMV infectionantiviral immunity T cell-mediated inflammation
INTRODUCTION
Adhesion molecules control cellular interactions within theimmune system and play a critical role in regulating leucocytehoming [1,2]. Thus, changes in the expression of adhesionmolecules on endothelial cells and circulating leucocytes areclearly important factors in controlling the formation of acellular exudate at sites of inflammation [3]. Recently, it hasbeen demonstrated that soluble isoforms of several adhesionmolecules are present in the circulation of normal individualsand may be increased during disease (for review see [4]), thusserving as diagnostic/prognostic markers. This is the case forICAM-1 (CD54), the expression of which is known to be up-regulated on many different cell types (including endothelialcells) by proinflammatory cytokines such as tumour necrosisfactor (TNF) and interferon-gamma [5-7]. Increased levels ofcirculating ICAM-1 (cICAM-l) have been reported in anumber of diseases, particularly such as are associated withsome kind of inflammatory reaction-infections, autoimmunediseases and cancer [8-13]. However, there have been fewlongitudinal studies of cICAM-1. This fact has contributedto the uncertainty as to whether the presence of cICAM-1 ismerely an early marker for inflammation or instead constitutes
Correspondence: A. Randrup Thomsen, Institute of MedicalMicrobiology, University of Copenhagen, The Panum Institute, 3CBlegdamsvej, DK-2200 N, Copenhagen, Denmark.
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a regulatory phenomenon contributing to the down-modula-tion of inflammation, whereby cICAM-l acts as a decoymolecule inhibiting continued leucocyte infiltration [4].
In the present study we have analysed the kinetics ofchanges in cICAM-1 levels associated with a systemic virusinfection using a well defined animal model, the murine lym-phocytic choriomeningitis virus (LCMV) infection, and wehave compared cICAM- 1 levels to other parameters ofimmune activation.LCMV is a non-cytopathogenic virus which causes little or
no non-specific inflammation. The specific antiviral immuneresponse, however, is associated with profound inflammatorychanges in infected organs, the appearance of which is depen-dent on CD8+ T cells [14]. Consequently, besides being amodel for systemic viral infections, results obtained in thissystem would also provide information of interest with respectto T cell-mediated inflammation in general.
In previous studies we have shown that LCMV infectioninduces marked polyclonal activation ofCD8 + T cells resultingin the generation of T cells with the phenotype CD IIahighCD49dhighCDl 1b+ CD62LIOw [15,16]. These cells are targetedfor homing to inflammatory sites, and this migration may beinhibited by MoAbs to CD1 la, CD49d or CR3 (CD1 lb,CD 18) [16-18]. In addition, the corresponding ligands includ-ing ICAM-1 have been found to be up-regulated on endothe-Hum and other cellular components in and near infectious foci
[16,18,19]. T cells play a critical role in this up-regulation, andbased on analysis of mice deficient in either MHC class I or
class II genes and consequently deficient in either CD8+ or
CD4+ T cells, it has been concluded that the presence ofCD8+ T cells suffices for induction of inflammation, whereasCD4+ T cells on their own induce less pronounced enhance-ment of ICAM-1 expression and a markedly reduced cellularexudate [19]. These findings are exploited in the present study,in which we have also studied the contribution of CD4+ andCD8 + T cells in mediating the changes in the level ofcICAM-1which may accompany a systemic viral infection.
anti-B220, were purchased from Pharmingen (San Diego, CA)as rat anti-mouse antibodies; anti-B220 was rat anti-mouseantibody from Cedarlane (Ontario, Canada).
Analysis of cICAM-1cICAM-1 in serum was quantified using a sandwich ELISA(Endogen, Boston, MA). The assays were run according to themanufacturer's instructions. Duplicate samples of each serumwere analysed, and median was calculated.
RESULTS
MATERIALS AND METHODS
MiceBALB/cA and BALB/cA-nu/nu mice were obtained fromBomholtgaard Ltd (Ry, Denmark). MHC class I-deficient(/32m -/-) mice, MHC class II-deficient (AO -/-) mice andcorresponding wild type littermates were all purchased fromGenPharm International (Mountain View, CA). Flow cyto-metric analysis of splenocytes from these mice revealed theexpected patterns of subset distribution [20-22]. Animals were
housed under controlled conditions that included testing ofsentinels for unwanted infections; no such infections were
detected. Female mice, 7-10 weeks old, were used in allexperiments, and the mice were always allowed to acclimatizeto the local environment for at least 1 week before use.
VirusLCMV of the Traub strain, produced and stored as previouslydescribed [23], was used in most experiments. In a few experi-ments, LCMV of the Armstrong strain and of the Aggressivevariant of the UBC strain were used; these strains were gifts ofM. B. A. Oldstone (Scripps Clinic and Research Foundation,La Jolla, CA), and of C. J. Pfau (Rensselaer PolytechnicInstitute, Troy, NY), respectively [24,25]. In addition, vesicularstomatitis virus (VSV) of the Indiana strain was employed; thisvirus was originally provided by K. Berg of this institute.Unless otherwise specified, mice received a virus dose of 103LD50 ofLCMV in an i.v. injection of 0-3 ml or an intracerebral(i.c.) injection of 0 03 ml. The i.v. infection results in transientimmunizing infection, whereas i.c. infection induces fatal T cell-mediated meningitis from which the animals succumb aroundday 8 post-infection (p.i.) [14,15].
Serum levels of cICAM-J in normal miceBased on analysis of serum samples taken from age-matchedcontrols in five independent experiments, we established thatthe median of the level of serum cICAM-1 in our BALB/cAmice was 12 3 pg/ml with a range 8-9-17 6 pg/ml (n = 16).
LCMV-induced changes in cICAM-J in serumTo study the effect of LCMV infection on cICAM-1 levels inserum, groups ofmice were infected with LCMV intravenously,and on each of the indicated days blood samples were takenfrom three animals, and serum was isolated and stored at -
20°C until analysis. The results of two independent experimentsare summarized in Fig. 1. It is evident that already on day 5 p.i.a significant increase in serum cICAM-1 level was observed inall animals tested. After this time point the level ofcICAM-1 inserum remained elevated for about a week, and on day 15 p.i.the level was back to normal.
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Flow cytometric analysisCells were stained with directly labelled MoAb, washed andfixed with 1% paraformaldehyde [15]. Immunofluorescencewas evaluated with a FACScan cytometer using the Lysys II
program (Becton Dickinson, Mountain View, CA). The follow-ing MoAbs were used in this study: PE-conjugated anti-CD4,FITC-conjugated and PE-conjugated anti-CD8a, PE-conju-gated anti-CD45R (B220), FITC-conjugated anti-CD la andFITC-conjugated anti-CD54 (ICAM-1). All antibodies, except
Fig. 1. Kinetics of changes in level of serum circulating ICAM-l(cICAM-1) in lymphocytic choriomeningitis virus (LCMV)-infectedmice compared with appearance of activated (CD ahigh) CD8+ Tcells. BALB/cA mice were infected intravenously with 103 LD50 ofLCMV, and on the indicated days serum samples were obtained andanalysed for cICAM- 1. Points represent individual mice; pooled resultsfrom two experiments are presented and different symbols denotedifferent experiments. Activated CD8 + T cells were quantified usingdirectly labelled anti-CD8-PE and anti-CDlla-FITC as previouslydescribed [16].
Additional experiments analysing cICAM-l levels in miceinfected with other stains of LCMV (Armstrong strain andAggressive strain) revealed essentially similar results (data notshown).Given that we have previously presented evidence indicating
that availability of activated CD8 + T cells is rate-limiting forthe generation of the virus-induced inflammatory response [18],the kinetics of the changes in cICAM-1 was compared toappearance of CD8 + CD 1 ahigh effector T cells in the spleen.It is clearly seen in Fig. 1. that the increase in cICAM-l inserum occurred before substantial clonal expansion of acti-vated T cells was observed.
Finally, to investigate whether the increase in serum cICAM-1 levels simply reflected shedding of ICAM-1 from the surfaceof activated T cells, ICAM-l expression on both CD4+ andCD8 + T cells and CD45R (B220) + B cells was analysed on day5 p.i. at which time almost peak levels of cICAM-1 areobserved. As can be seen in Fig. 2., some increase in ICAM-1expression was observed for all lymphocyte subsets at this time,suggesting that the increase in cICAM-I level was the result of amore general increase in ICAM-1 expression.
LCMV-induced increase in cICAM-J precedes cellularinfiltrationAs mentioned above, the results presented in Fig. 1. suggestedthat the increase in cICAM-l would precede the formation ofthe cellular exudate in infected organs. However, to study thismore directly, a group of BALB/cA mice were infected withLCMV intracerebrally, and on days 3, 5 and 7 p.i. the level ofcICAM-I in serum was assayed in three mice as was themeningeal inflammation in the same animals. From the resultspresented in Fig. 3, it is evident that the level of cICAM-1 inserum was markedly increased before substantial cellular infil-tration into the meninges could be observed.
Role of T cells in the LCMV-induced increase in cICAM-1Although T cell activation can be detected as early as day 3-5p.i. using other methods [16], the finding that elevation ofcICAM-1 level in serum took place before substantial clonalexpansion of activated T cells could be demonstrated (Fig. 1)made it pertinent to ask whether the increase in cICAM- I levelsin fact occurred independently of T cells, e.g. as a result ofinterferon production or natural killer (NK) cell activation. Toanswer this question, T cell-deficient nu/nu mice were infected
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in parallel to wild type controls, and on days 5, 8 and 15 p.i. thelevel of cICAM- 1 in serum was assayed in three mice of eachphenotype. At no time point did we observe a significantincrease in serum cICAM-1 in nude mice (Fig. 4), indicatingthat T cells were mandatory for LCMV infection to induce anincrease in cICAM-1 levels in serum.
In order to evaluate the role ofT cell subpopulations in thisresponse, we next infected groups of MHC class I and MHCclass 11-deficient mice which are lacking CD8 + and CD4+ Tcells, respectively [20-22]. Analysis of serum samples taken onday 6 p.i. revealed that at least the same increase in cICAM-1level was observed in class II-deficient mice as in wild typecontrols (Fig. 5); this is consistent with our previous findingthat LCMV-induced inflammation is unimpaired in thesetransgenics [19,26]. However, we also found a substantialincrease in cICAM-1 level in two out of three class I-deficientmice tested, suggesting that CD4+ and CD8 + T cells indepen-dently suffice for serum cICAM-1 levels to be increased byLCMV.
VSV-induced changes in cICAM-I in serumFinally, to investigate whether a virus causing less pronouncedT cell activation would lead to similar changes in cICAM-1level in serum, mice were infected with 2 x 107 plaque-formingunits (PFU) ofVSV intravenously. VSV is not a natural mousepathogen and viral replication is limited [27]. However, asubstantial innate immune response is observed, and aroundday 6 p.i. a cytotoxic T cell response can be detected [27-29]. Ascan be seen in Fig. 6, a marked increase in serum cICAM- 1level was found on day 1 p.i., probably reflecting activation ofinnate immune defence mechanisms. More important in thiscontext, the cICAM-1 level remained significantly elevated ondays 3-7 p.i., which coincide with the expected period ofT cellactivation as previously described [29].
DISCUSSION
This study demonstrates that a soluble form of ICAM-1 isreleased in increased amounts into the serum of virus-infectedmice. In the case of LCMV infection, the resulting increase inthe level of serum cICAM- 1 was found to require the presenceof T cells, as no increase was observed in infected T cell-deficient mice. Notably, high levels of cICAM-1 in serumwere found well before maximal T cell activation was observed;
Fig. 2. Expression of ICAM-1 on CD4+ (a), CD8+ (b), and CD45R (B220)+ B cells (c) on day 5 p.i. Splenocytes were stained withdirectly labelled anti-CD4-PE, anti-CD8-PE, or anti-B220-PE and anti-ICAM-1-FITC; gates were set for PE-positive population.Splenocytes from uninfected controls were analysed in parallel. Curves are representative of at least three animals.. , Control;
, infected.
O 1995 Blackwell Science Ltd. Clinical and Experimental Immunology, 102:268-273
Fig. 3. The increase in circulating ICAM-1 (cICAM-l) level in serumprecedes the formation of the cellular exudate. BALB/cA were infectedwith 103 LD50 of lymphocytic choriomeningitis virus (LCMV) intra-cerebrally, and on the indicated days serum samples from three animalswere analysed for cICAM-1 concentration, and cerebrospinal fluid(CSF) from the same animals were analysed for cellular infiltration.Medians and ranges are presented.
thus elevation of serum cICAM-1 coincided with the earliestevidence of T cell activation [16]. Our results on the kinetics ofthe LCMV-induced changes therefore support the idea that anincrease in cICAM-1 in serum is an early marker of immuneactivation rather than the alternate hypothesis, namely thatrelease of cICAM-1 is a way in which down-regulation ofinflammation is accomplished. The latter has been a distinct
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Fig. 5. Serum circulating ICAM-1 (cICAM-l) levels in lymphocyticchoriomeningitis virus (LCMV)-infected MHC class I and MHC classII-deficient mice. MHC class I (CID) and class II (C2D)-deficient miceand wild type littermates (wt) were infected with 103 LD50 of LCMV,and on day 6 p.i. serum concentration of cICAM-l in these mice andsham-inoculated controls (n) was determined. Points represent indivi-dual mice.
possibility because cICAM- I has been shown to be functionallyactive in binding to its leucocyte ligand, LFA-1 [30,31], andthus potentially could block leucocyte-endothelial cell interac-tions critical to leucocyte extravasation.
As to the mechanism underlying the increase in serumcICAM-1 levels in LCMV-infected mice, we could findincreased expression of ICAM-1 on both T cells and B cellsearly in the infection. This, and the fact that an increase inserum cICAM-1 could be observed also in mice deficient in
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Fig. 4. The lymphocytic choriomeningitis virus (LCMV)-inducedincrease in circulating ICAM-1 (cICAM-l) level in serum requires Tcells. T cell-deficient nu/nu (0) and normal BALB/cA mice (0) were
infected with 103 LD50 of LCMV intravenously and on the indicateddays serum cICAM-1 level was determined in three mice per group.
Points represent individual mice.
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Fig. 6. Kinetics of changes in level of serum circulating ICAM-1(cICAM-l) in vesicular stomatitis virus (VSV)-infected mice. BALB/cA mice were infected intravenously with 2 x 107 plaque-forming units(PFU) of VSV, and on the indicated days serum samples were obtainedand analysed for cICAM-l. Points represent individual mice.
() 1995 Blackwell Science Ltd, Clinical and Experimental Immunology, 102:268-273
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CD8 + T cells, where few activated T (CD4+) cells are present[26], indicate that while T cells are mandatory, their role israther indirect. In parallel to the observed increase in cICAM- Ilevels in class I-deficient mice, we have previously found thatalthough effector T cell generation and the resulting T cell-dependent inflammatory exudate was markedly reduced inthese mice, significant up-regulation of endothelial expressionof ICAM-l could be seen in infected organs [19]. Therefore, wepropose that cICAM-1 in serum is a sensitive marker forcytokine release and endothelial activation for which a limitednumber of activated T cells seem to suffice. That only few Tcells should be required to induce marked endothelial activa-tion seems physiologically reasonable, as it would reduce thelikelihood of infected cells evading detection by circulatingeffector cells passing by. Moreover, this explanation would fitwith the finding that an increased level of cICAM-I in serum isobserved already early in the infection before the bulk of acti-vated T cells are generated, and also that essentially the sameinitial pattern is observed in mice deficient in CD8 + T cells.As to the practical implications of our findings, it is impor-
tant to note that a significant increase in serum cICAM- 1 couldbe detected in all LCMV-infected animals tested on day 5 p.i.Given that we have previously shown that immunosuppressivetreatment started on this day could completely inhibit the Tcell-mediated inflammatory response [32], the present resultsindicate that it would be possible to initiate treatment of thisimmune-mediated disease based on monitoring of cICAM-llevels in serum. While this in itself is of little consequence, itsuggests that a similar approach might be used in cases where Tcell-mediated inflammation is unwanted, e.g. in the context ofallograft transplantation or autoimmune disease.
ACKNOWLEDGMENTS
This work was supported in part by the Danish Medical ResearchCouncil, Grant 12-1482, the Novo Foundation, the Beckett Founda-tion, the Association for Rheumatoid Arthritis, and the BiotechnologyCentre for Signal Peptide Research. J.P.C. is the recipient of a PhDscholarship from the Faculty of Health Sciences, University of Copen-hagen.
NOTE ADDED IN PROOF
Further supporting a role for cICAM-1 as marker for virus-induced inflammation, we have recently found a markedlyincreased level of cICAM-1 in CSF of mice infected intracere-brally with LCMV (l 14 Mg/ml versus < 0 1 Ig/ml in controls).
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