Evidence for Prostaglandin-ProducingSupressor Cells in HCV Patients with

Normal ALTRUI MARINHO, MD, RUTH PINTO, MS, MARIA LIVIA SANTOS, BS, and

MIGUEL CARNEIRO DE MOURA, PhD

Our objective was to verify the presence of prostaglandin-producing suppressor cells inresponse to hepatitis C virus antigens in peripheral blood mononuclear cells proliferation.Standard proliferation tests were performed in 31 patients: 20 with chronic hepatitis C afterantiviral treatment [7 long-term responders (LTR), 7 relapsers (RR), 6 nonresponders (NR)],7 with HCV infection with persistently normal aminotransferase levels (PNAL), and 4 withhepatocellular carcinoma. Six antigens were used from the core and NS3 regions. A modifiedproliferation assay consisting of the addition of indomethacin was also done. Lymphoprolif-erative responses to the HCV antigens were detectable in 27% (11/41) of test points of LTR,10% (3/31) of RR, 26% (9/35) of NR, and 18% (7/39) of patients with PNAL. Indomethacinonly had effect in PNAL patients, by increasing the frequency of reactivity from 18% (7/39)to 36% (14/39) tests points (P � 0.037); also, in three of these patients (43%) indomethacinstrongly modified proliferation to core 31–50 and NS3 1248–1261 antigens, increasing boththe frequency and stimulation index from 33% (6/18) to 72% (13/18) (�2 � 5.43, P � 0.019)and 1.89 � 0.43 to 6.18 � 4.74 (P � 0.028), respectively. These results suggest thatprostaglandin-producing suppressor may play a role in chronic HCV infection by inhibitingcellular immune responses in patients with persistently normal ALT.

KEY WORDS: hepatitis C; aminotransferases; hepatocellular carcinoma; T-lymphocytes; indomethacin.

Hepatitis C virus infection is a leading cause ofchronic hepatitis, cirrhosis (1), and hepatocellularcarcinoma (2), with significant morbidity and mortal-ity. It is estimated that 170 million people, approxi-mately 3% of the world’s population, are infectedwith this virus (3). Most people (�85%) infected withHCV develop chronic infection, despite being able togenerate virus-specific antibody (4–6) and T cell re-sponses (7–9). Considerable interest has been focusedon the possible reasons for the high rate of viral

persistence (10). One explanation for this nonprotec-tive immunity could be the emergence of variants thatescape humoral and cellular immune responses (11).

Cellular immune responses play an important rolein the immunopathogenesis of chronic HCV infectionand thus affect its outcome (12). Lymphocyte prolif-erative responses to HCV antigens have been de-tected in peripheral blood of patients with chronicHCV infection, but a clear relationship with diseaseactivity can not always be found. In contrast withpatients having acute self-limited hepatitis C (13, 14),which shows a strong T helper response to NS3,responses in patients with chronic infection are notalways easily detected (15, 16).

Low sensitivity or absence of reactivity in vitro ofperipheral blood mononuclear cell assays could be

Manuscript received May 14, 2001; accepted August 29, 2001.From the Liver Unit, Centre of Gastroenterology, Hospital

Santa Maria, Medical School of Lisbon, Portugal.Address for reprint requests: Dr. Rui Tato Marinho, Hospital

Santa Maria, Medicina 2, Av. Prof. Egas Moniz, 1649-028 Lisbon,Portugal.

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556 Digestive Diseases and Sciences, Vol. 47, No. 3 (March 2002)0163-2116/02/0300-0556/0 © 2002 Plenum Publishing Corporation

the result of various mechanisms, such as the lowfrequency of specific T cells in cultures, compartmen-talization of some CD4� cells in the inflamed liver, orsequence heterogeneity among HCV isolates used invitro compared to those from patients (17).

Another less explored mechanism is the possibilitythat prostaglandin-producing adherent suppressorcells could be causing low reactivity by modulating theimmune response and inhibiting the lymphocyte pro-liferative response to HCV antigens. It is now recog-nized that the lymphocyte proliferation response toan antigen can be influenced by the presence of anincreased suppressor cell activity mediated by glass-adherent monocytes producing prostaglandin E2(PGE2). PGE2 is widely viewed as a general immu-nosuppressant and is known to inhibit IL-2 and IFN-�production from T helper cells, T-cell cytotoxicity,natural-killer and antibody-dependent cytotoxicityand to decrease the responsiveness of T cells tomitogens (18, 19).

The presence of prostaglandin-producing adherentsuppressor cells has already been described in otherimmunological situations, some of them chronic in-fections like tuberculosis, (20) schistosomiasis man-soni (21), drug-induced liver injury (22), Hodgkin’sdisease (23), Crohn’s disease (24), and sarcoidosis(25).

In the present study we used a modified prolifera-tion assay of peripheral blood mononuclear cells(PBMC) in the presence of a prostaglandin inhibitor(indomethacin) in order to assess the presence ofadherent suppressor cell activity in hepatitis Cchronic infection.

MATERIALS AND METHODS

Patients and Controls. PBMC studies were performed on31 patients with different courses of HCV infection (Table1). Among these patients there were 20 with chronic hep-atitis C after treatment with interferon: 7 long-term re-sponders (LTR), defined by having normal ALT and neg-ative HCV-RNA by PCR for one year after stoppingtreatment (average follow-up period was 46.7 months,

range 18–84 months); 7 with biochemical response duringthe treatment but relapsing after the suspension of inter-feron (RR); and 6 nonresponders (NR). Four patients withhepatocellular carcinoma (HCC) were also evaluated.

We also included a group of seven patients with chronicHCV infection, sometimes called “HCV healthy chroniccarriers,” having persistently normal ALT levels (PNAL).They were followed for an average of 7.8 years (range 2–16years). For this study we defined PNAL as repeatedlynormal levels of serum AST and ALT on at least threeconsecutive occasions over a period of six or more consec-utive months. In this group the HCV RNA was persistentlynegative in three patients, oscillating, ie, negative to posi-tive at low levels (0.2–0.3 � 106 meq/ml) in other three, andpositive in only one of them at 2.0 � 106 meq/ml. Fourconsented to liver biopsy: two showed normal liver and 2showing mild chronic hepatitis, in terms of activity andfibrosis.

We also studied eight healthy controls (AST and ALTnormal, negative for HBsAg, HIV, and anti-HCV, withoutalcohol consumption).

All patients and controls gave informed consent to thestudies according to the ethical guidelines of the HelsinkiConference. The Ethical Committee of the Medical Facultyof Lisbon approved the protocol.

HCV Antigens. HCV antigens consisted of six purifiedrecombinant HCV peptides, kindly provided by HelmutDiepolder and Gerd Pape (Institut fur Immunologie, Uni-versitat Munchen, Munich, Germany), from core [aminoacids (aa) 21–40, 31–50, 141–160] and NS3 regions (twodifferent antigens from aa 1248–1261 and one from aa1388–1409).

PBMC Proliferation Assay. PBMC were separated fromheparinized venous blood, washed three times in phos-phate-buffered saline (PBS) and resuspended at a concen-tration of 1 � 106 cells/ml in RPMI; 100 �l of PBMC werecultured in triplicate in a 96-well microtitre plate (Costar�)in the presence of 2 �g/ml of HCV antigens. Cells werecultured for six days at 37°C in a humidified, 5% CO2-enriched atmosphere. Sixteen hours before the end of theculture period, [3H] thymidine was added, and the rate ofincorporated radioactivity (counts per minute) was deter-mined by liquid scintillation counting in a �-counter.

Results are expressed as percentages (the sum of tests inwhich proliferation was observed/total number of tests per-formed for each group) and stimulation indexes (SI �counts per minute of cultures with HCV antigen/counts perminute of cultures without HCV antigen). Medium andPHA (phytohaemaglutinin)/PWM (pokweed mitogen)-

TABLE 1. GROUPS OF PATIENTS AND CONTROLS STUDIED

Patients NGender

F/M Age (yr)

HCV RNA(�106

meq/ml)

Long-term responders 7 2/5 42.0 (33–49) 0Response and relapse 7 1/6 44.8 (35–62) 1.10 (0.31–2.3)Nonresponders 6 3/3 47.5 (25–58) 1.86 (0.25–3.3)Hepatocelular carcinoma 4 0/4 69.2 (65–74) 1.1 (0–3.0)PNAL 7 6/1 37.4 (21–50) 0.43 (0–2.0)Controls 8 6/2 38.7 (24–55) 0

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induced PBMC served as culture controls. Results wereconsidered positive when SI was higher than 2.5.

Modified Proliferation Tests in Presence of Prostaglan-din Inhibitor. A modification to the lymphocyte prolifera-tion assay, consisting of the addition of a prostaglandininhibitor (PI; indomethacin 1 �g/ml final concentration) tothe cultures stimulated with the HCV antigens, was simul-taneously used for patients and controls. Several studieshave shown that it is possible to restore the proliferativeresponse by adding prostaglandin inhibitors to the cultures(26).

None of the controls had SI � 2.5 in response to any ofthe HCV recombinant antigens.

HCV RNA. Serum HCV RNA was detected by using theCobas Amplicor version 2.0 HCV polymerase chain reac-tion (PCR) kit (Roche Molecular Systems, Basel, Switzer-land).

Statistical Analysis. Frequency of proliferation and stim-ulation indexes before and after indomethacin were com-pared by the �2 test and the Wilcoxon signed ranks test, andwere considered significant if P � 0.05.

RESULTS

Without indomethacin, significant HCV-specificcellular immune reactivity was detected in 27% oftests done in the LTR (11/41), 10% in RR (3/31),26% in NR (9/35), 18% (7/39) in PNAL patients, andin none with hepatocellular carcinoma (0/20). Theaverage SI of the positive tests in each category wereas follows: 4.6 � 2.1 for LTR, 4.8 � 1.4 for RR, 6.8 �3.9 for NR, and 5.2 � 1.8 in PNAL group; P � NS.

The addition of indomethacin did not significantlyincrease the percentages of positive tests and the SI inthe groups of patients with LTR, RR, NR, or HCC.(Table 2). In contrast, after adding indomethacin tothe group of patients with PNAL, we observed asignificant change in the lymphoproliferation. Thepercentage of positive tests (the number of tests inwhich proliferation was observed/total number oftests performed for each group) increased from 18%(7/39) to 36% (14/39) (P � 0.037, Wilcoxon signedrank test) (Figure 1).

In three of the PNAL patients the change of thepercentage of positive tests and the SI values afterindomethacin addition pointed to greater CD4� re-activity: the frequency of positive wells increasedfrom 33% (6/18) to 72% (13/18) (�2 � 5.43, P �0.019). In the same patients, proliferative responsesto two specific antigens, core aa 31–50 and NS31248–1261 previously negative, always became posi-tive after adding indomethacin, showing an increasein the SI from 1.89 � 0.43 to 6.18 � 4.74 after theaddition of the prostaglandin inhibitor, P � 0.028(Figure 2).

In the patients with PNAL who showed a strongincrease in the proliferation after indomethacin, wefound lower HCV RNA levels (0.12 � 106 meq/ml)and no histopathologic lesions two had a normalliver) when compared to those without response(1.8 � 106 meq/ml of HCV RNA (P � NS), two withmild chronic hepatitis).

DISCUSSION

In vitro T-cell proliferative responses to hepatitisvirus-encoded proteins lack sensitivity and are notalways detected. These disadvantages could be theresult of the role played by suppressor cells in asso-ciation with altered humoral and cellular immunity.Prostaglandin-producing suppressor cells have beendescribed in the peripheral blood of normal humans(27). These cells, presumably monocytes (28, 29), areglass-adherent and secrete PGE2, which suppressesthe response of T cells to mitogens in vitro. PGE2 canbe considered an important regulatory modulator ofthe immune system for its inhibition of mitogen-stimulated human T-cell proliferation, inhibition ofproduction of IL-1, and TNF-� by mouse macro-phages (30), IL-2, IFN-� (31), as well as for its sup-pression of human natural killer cell activity (32).Activated monocytes in cultures of PBMC can secrete

TABLE 2. FREQUENCY OF POSITIVE TESTS (POSITIVE WELLS) AND STIMULATION INDEXES (SI) WITHOUTAND WITH INDOMETHACIN

Patients

Lymphocyte proliferation

P

Without indomethacin With indomethacin

N � 183 SI N � 183 SI

Long-term responders 27% (11/41) 4.6 � 2.1 22% (9/41) 4.5 � 3.2 NSResponse and relapse 10% (3/31) 4.8 � 1.4 3% (1/31) 3.1 NSNonresponders 26% (9/35) 6.8 � 3.9 23% (8/35) 7.0 � 2.6 NSHepatocellular carcinoma 0% (0/20) 0% (0/20)PNAL 18% (7/39) 5.2 � 1.8 36% (14/39) 5.5 � 3.1 0.037*

*Wilcoxon signed ranks test for the percentages of positive tests without and with indomethacin.

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prostaglandins, namely PGE2 (known to have imuno-modulatory effects by inhibition of helper T cells orstimulation of suppressor T cells) that inhibit, assuppressor cells, lymphocyte proliferation.

In our work, the addition of indomethacin in-creased the sensitivity of the test by reversing theeffect of suppressor cells. This same effect has alsobeen found in other situations, such as tuberculosisand schistosomiasis (33). Indomethacin increased thefrequency of CD4�-specific HCV responses in thegroup of patients with persistently normal ALT levelsonly (healthy chronic carriers), by increasing the fre-

quency of Th responses from 18% (7/39) to 36%(14/39) tests points (P � 0.037, Wilcoxon signedranks test). We did not obtain similar results in theother groups of patients.

In 43% (3/7) of the PNAL patients, indomethacinstrongly modified the specific HCV proliferation totwo antigens, (core 31–50 and NS3 1248–1261) byincreasing both the frequency and the stimulationindexes from 33% (6/18) to 72% (13/18) (P � 0.037),and from 1.89 � 0.43 to 6.18 � 4.74 (P � 0.028),respectively.

The activity of the suppressor cells in patients withchronic HCV infection, without significant liver dam-age and normal ALT (chronic carriers?), could beresponsible for the negative modulation of the im-mune response, and so decrease the liver damage.Several lines of experimental work point to the factthat it is the immune response that leads to liverinjury and not the virus, which is not cytopathic (34).

The presence of prostaglandin-producing cells wasmore evident in response to two highly conservedregions of the genome, one from the core (core aa31–50) and the other from an epitope with a length of14 amino acids in the helicase region (NS3 aa 1248–1261). The proliferative responses were previouslynegative in all and became positive in 43% (3/7) ofthe PNAL patients and in none of the other 21belonging to other groups (�2 � 7.92, 0.005, Fisherexact test). Other authors have also shown strong

Fig 1. Stimulation indexes in the different groups of patients. Numbers in parenthesisrepresents the number of tests done for each group. A; without indomethacin, B; afteradding indomethacin. *P � 0.037 (Wilcoxon signed ranks test).

Fig 2. Stimulation indexes in patients with normal ALT respondingto indomethacin, regarding HCV antigen core aa 31–50 and NS31248–1261. *P � 0.028 (Wilcoxon signed ranks test).

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HCV-specific proliferative responses to these regions(35–37). This response is of particular interest be-cause in general the CD4-mediated response is mul-tispecific, but immunodominant epitopes, like theNS3 sequence 1248–1261 (38, 39), have been identi-fied by studying the specificity of HCV-reactive poly-clonal T cell lines derived from PBMC stimulationwith recombinant HCV proteins (40). Our worktaken together with that of other authors indicatesthat these sequences, highly promiscuous and locatedwithin a highly conserved HCV region, could there-fore be a promising candidate for the design of pre-ventive or immunotherapeutic vaccines.

Our results also suggest a possible role of suppres-sor T cells producing PGE2 in the immunopathogen-esis of HCV infection, especially in patients withpersistently normal ALT. These findings may haveimplications for the design of new treatment strate-gies for HCV patients with normal serum ALT whohave poor responses to interferon. A recent study inpatients with chronic hepatitis C treated with oralprostaglandin (PGE2) has shown that the medianlevel of HCV RNA decreased during the treatmentperiod, although this decrease was not sustained (41).The association of interferon to NSAIDs in this par-ticular group, having normal ALT, warrants furtherinvestigation.

ACKNOWLEDGMENTS

We thank to Alice Restolho, Celeste Costa, and PreciosaBastos for technical assistance in the laboratory work.

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