AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 7, 1992 Mary Ann Liebert, Inc., Publishers

Hyperimmunoglobulinemia in HIV-1 Infected Individuals Does Not Clearly Correlate with Plasma Levels of IL-6 MARIA CARLA RE,1 GIORGIO ZAULI,1 GIULIANO FURLINI,1 MONICA VIGNOLI, ERIC RAMAZZOTTI,1 SERGIO RANIERI,2 and MICHELE LA PLACA1

ABSTRACT In this study we evaluated interleukin-6 (IL-6) plasma levels in 80 human immunodeficiency virus type 1 (HIV-1) seropositive ( + ) individuals and 51 HIV-1 seronegative (— ) blood donors. Plasma IL-6, detectable only in a subset of HIV-1( + ) individuals (45 of 80) and normal blood donors (28 of 51), was significantly (p < 0.01) increased in HIV-1( + ) subjects 187 ± 20.5 vs. 86.3 ± 14 pg/ml). Among HIV-1-infected individuals, ARC/ AIDS patients showed the highest IL-6 values (243.3 ± 43.3 pg/ml). HIV-1( + ) subjects showed, at all the different stages of the disease, a significant increase in total gammaglobulins, particularly IgG (2071 ± 101 vs 1265 ± 34 of HIV-1 seronegative controls). Although among HIV-1-infected individuals, the group with detectable plasma levels of IL-6 shows the highest levels of IgG (2243 ± 146 vs. 1790 ± 105, < 0.05), no positive correlations were observed between plasma levels of IL-6 and total gamma globulins (r 0.2) or IgG (0.17). IL-6 production was also examined in the endotoxin-free supernatants of peripheral blood cultured monocytes and CD4+ lymphocytes, in the presence or absence of specific stimuli. The amount of IL-6 released in monocyte and CD4+ T-lymphocyte culture supernatants was similar in 40 HIV-1( + ) individuals and 35 HIV-1() controls. =

Our data show that plasma levels of IL-6 are significantly increased in HIV-1-infected individuals, in particular in ARC/AIDS patients. However, such an increase does not strictly correlate with the degree of hypergammaglobulinemia in the same HIV-1-infected individuals.

INTRODUCTION

I

NTERLEUKIN-6 (IL-6) titude of biological

IS A

pleiotropic cytokine.

effects:1-3 it regulates

with a mulimmune re-

sponses,4-6 acute phase proteins,7 and hematopoiesis8y playing crucial role in host defense mechanisms. One main target of IL-6 is the immune system through induction of B-cell differentiation into antibody-forming cells, '" induction of IL-2 and IL-2 receptor expression," proliferation and differentiation in T-cells.5'2 An increased production of IL-6 has been implicated in polyclonal B-cell activation with autoantibody production and autoimmune diseases.13 Polyclonai B-cell activation is also characteristically seen in various a

stages of human immunodeficiency virus-type 1 (HIV-1) infec¬ tion and AIDS: hypergammaglobulinemia, an increase in Igsecreting cells, the presence of autoantibodies and an elevated frequency of cell lymphomas.'4~'ft It has recently been reported that HIV-1 induces RNAm expression of IL-6 in normal PB mononuclear cells'7 and that IL-6 plasma levels are elevated in HIV-1-infected individuals. ·]i> IL-6 production has been related to HIV-1 replication in monocytes and HIV-1 strains with a selective tropism toward '8 monocytes induce IL-6 production most efficiently. Nevertheless, other in vitro and in vivo reports failed to confirm that HIV-1 infection raises IL-6 production by PB monocytes,20·21 which are the major cellular source of IL-6.

'Institute of Microbiology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy. 2"Santa Maria delle Croci" Hospital, Ravenna, Italy. 1289

1290

RE ET AL

In this study we investigated the IL-6 plasma levels in 80 HIV-1 + individuals at different stages of the disease (WRI to WRVI) in comparison with 51 HIV-1 blood donors. In a subset of HIV-1-infected individuals (40 subjects) and HIV-1 blood donors (35 subjects) IL-6 production was also determined in the supernatants of PB CD4+ T-cell and monocyte short-term cultures.

MATERIALS AND METHODS Patients and controls +

Peripheral

blood samples were collected from 80 H1V-1 individuals and 51 HIV-1 blood donors, who previously gave their written informed consent to this research, according to the Helsinki declaration. Most HIV-1+ subjects (36 Classes I-IL 17 III-IV, 27 V-VI of Walter Reed classification), 60 males and 20 females, were studied before any administration of antiretroviral therapy with azidothymidine or other drugs. None of them displayed intercur¬ rent illness at the time of the study. They showed a mean number of 475 ± 41 CD4+ lymphocytes and 1005 ± 69 CD8+ with CD4/CD8 ratio of 0.47 ± 0.04, Hb a lymphocytes, 13.4 ± 2.3 g/100 ml, white blood cell count 5620 ± 282 µ , ~

platelets 204,000

±

13,000/µ1. comprised sex-

Normal controls HIV-1 blood donors.

and

age-matched healthy

-

Sample preparation Plasma samples were diluted 1/1 with sterile saline and frozen 70°C within 2-3 hours since blood taking. No IL-6 assays were performed on any plasma samples that had been frozen and thawed more than once. Plasma samples were usually heatinactivated to avoid IL-6 binding to A2M, a serum IL-6-binding carrier protein, which could interfere with the immunoenzy¬ matic assay, blocking the immunoreactive epitopes of IL-6. However, no significant differences in the level of IL-6 by the immunoenzymatic determination were noticed before and after heat-inactivation (data not shown). In 40 HIV-1 + individuals and 35 HIV-1 blood donors, the production of IL-6 was also investigated in the supernatant of different short-term cultures of PB populations. Herparinized (20 U/ml) PB specimens were diluted 1/2 with Iscove's modified Dulbecco's medium (IMDM, Gibco. Grand Island, NY), supplemented with penicillin (250 U/ml) and at

—

streptomycin (250 µg/ml), layered over Ficoll-Histopaque 1.077 g/ml, Pharmacia, Uppsala, Sweden) and centri¬ („ fuged at 1500 rpm for 30 min. Light-density mononuclear cells were collected and washed twice in IMDM supplemented with =

10% fetal calf serum (FCS). Monocytes were obtained by incubating light-density mono¬ nuclear cells (2-3 x I06 cells/ml) in plastic flasks with IMDM + 10% FCS at 37°C in a fully humidified atmosphere of 5% CO, in air for 1 h. Mononuclear nonadherent cells were decanted, and monocytes collected by vigorous flushing with cold medium. This cellular fraction contained about 90% esterase+ cells and CD14+ cells, when determined by nonspecific esterase staining and by immunofluorescence staining with

FITC-conjugated anti-CD14 (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Enriched lymphocytes were obtained from mononuclear nonadherent cells by two successive steps of rosetting with fresh neuraminidase-treated red blood cells. After two washings with NH4CI in order to lyze red blood cells, cells were seeded at 5

10'' cells/ml in round bottom tubes in the presence of

x

immunomagnetic beads, coated with an anti-CD4 antibody (M450. Dynabeads, Dynal, Oslo, Norway) in 300 µ of cold IMDM + 1% BSA, for 30 min in ice, under continuous agita¬ tion. An immunomagnetic beads/cells ratio of 3:1 provided the best recovery of CD4+ lymphocytes. CD4+ lymphocytes coated with immunomagnetic beads were recovered by a magnet (MPC1. Dynal) and resuspended ini ml of IMDM + 10% FCS. After overnight incubation, cells were gently pipetted 25-30fold to facilitate cell detachment from immunomagnetic beads, which were eliminated by a second passage in the magnet. This enriched CD4+ T-cell preparation contained 96-97% CD4+ cells, less than 1% monocytes (CD14+), lymphocytes (CD20+), and NK (CD16 ) detected by immunofluorescence staining with FITC-conjugated anti-CD3, anti-CD14, anti +

CD-20 and anti-CD16 monoclonal antibodies (Becton Dickin¬ son) and nonspecific esterase staining.

HIV-1 isolation, HIV-1 p24 transcriptase (RT) assay

antigen detection,

reverse

For HIV-1 isolation, 2 x 106 PB patient light-density mono¬ nuclear cells were cocultured with 1 x 106 normal PB lightdensity mononuclear cells, pretreated for 72 h with 1 pg/ml of PHA. Cultures were maintained in 3 ml IMDM + 10% FCS, IL-2 (10 U/ml Sigma, St. Louis, MO), anti-IFN-a (Amersham, 1,870 U/ml) and polybrene (Sigma) 1 µg/ml. Culture superna¬ tants were tested twice weekly both for the presence of p24 core protein and for the presence of detectable levels of RT, as

previously described.22 In vitro cultures

lymphocytes

of PB-enriched monocytes and CD4+

In preliminary experiments, optimal stimulating concentra¬ tions of bacterial lipopolysaccharide (LPS from Eschericha coli, Sigma, 1 pg/ml), phytohemagglutinin (PHA, 10 µg/ml), and

acetate (PMA, 10~'°M) were established shown). Monocytes were cultured for 48 h in IMDM plus 10% FCS at a concentration of 1 x 106 cells/ml in the absence or presence of 1 µg/ml of LPS. CD4+ lymphocytes were cultured at a concentration of 1 x 10'7ml in IMDM plus

phorbol myristate (data

not

10% FCS in the absence or presence of 10 pg/ml PHA plus 10"' M PMA for 72 h. At the end of the incubation period, supernatants were harvested and passed through 0.45 µ pore size filters, then stored at —80°C until cytokine determination was

performed.

Assay for IL-6 activity Various dilutions (undiluted samples and samples diluted 1:2, 1:4, 1:8) of plasma samples and culture supernatants from unstimulated and stimulated enriched monocytes and CD4+ lymphocytes were assayed by an ELISA kit for the specific quantitative determination of natural human IL-6 (R&D Sys-

PLASMA LEVELS OF IL-6 IN HIV-1+ SUBJECTS

pg/ml). The kit specifically native or recombinant human IL-6 with no detectable cross reaction with other cytokines. In some experiments, to exclude the possibility that the immunoenzymatic determination could not adequately detect plasma levels of IL-6 because of the interference with serumbinding proteins, the levels of plasma IL-6 were also determined with a standard biological assay, utilizing the IL-6-dependent B9 cell line.23 The results were similar to those obtained with the immunoenzymatic determination. In particular, 3 HIV-1+ indi¬ viduals, who presented undetectable levels of plasma IL-6 with the immunoenzymatic determination, were completely unable to stimulate the proliferation of B9 cells (data not shown). terns, lower limit of detection 15 measures

Statistical

analyses

The results are expressed as mean ± SEM of the data ob¬ tained from at least three separate experiments performed in duplicate. Pearson's test for correlation (r), chi-square test, and two-tailed Student's r-test for unpaired data were used for statistical comparison between HIV-1 samples and normal controls.

RESULTS Plasma IL-6 levels in The

mean

values of

HIV-I-infected subjects plasma

IL-6 showed

significant

a

+

0.01) increase in 80 HIV-1 individuals compared with 51 HIV-r blood donors (187 ± 20.5 vs 86.3 ± 14 pg/ml).

(p


0)

l» -

--

+ Normal blood donors

44W.R. V-VI W.R. l-ll W.R. III-IV H)v_i Séropositive subjects

FIG. 1. Immunoenzymatic determination of plasmatic levels of IL-6 in 51 normal blood donors ( ) and 80 HIV-1 seropositive individuals ( ), divided in three groups according to the Walter Reed Classification (WR I-II, WR III-IV, WR V-VI). Horizontal bars: mean values of each group of subjects.

1292

RE ET AL BOOO

E

of HIV infection."419 We actually found that, among HIV-1 + individuals, the mean IgG level is higher in patients with detectable plasma levels of IL-6 than those with undetectable plasma levels of IL-6 (2243 ± 146 IgG vs. 1790 ± 105 mg/dl,

5000

0.05) (Fig. 2). positive correlations were observed between plasma levels of IL-6 and total gammaglobulins (r 0.2. 0.45) or IgG (r 0.17. chi-square 3.06, chi-square 3.15, 0.5) (Fig. 3). Moreover, at variance with plasmatic levels of IL-6 that were particularly increased in ARC/AIDS patients. IgG levels were

respectively,

O

However,




._

lymphocytes).

Since the production of IL-6 has also been related to an active HIV-I replication in monocytes,18 IL-6 levels in the plasma and culture supernatants were correlated with the ability of patient's PB cells to infect normal PB leukocytes, as judged by the appearance of p24 antigen and reverse transcriptase activity in the supernatants of cocultures. No significant correlations were observed between the ability to infect normal PB LD-MC and IL-6 levels either in plasma (r 0.35) or in LD-MC (r 0.22) and monocyte (/' 0.31 ) culture supernatants (data not shown).

2000-

=

=

=

E I

a

a.

1000--

o-1-

4-

100

4-

200

4-

300

4-

400

500

-¬ 600

DISCUSSION 700

Plasmatic IL-6 levels in HIV-1 seropositive subjects FIG. 4. IgG plasmatic levels in 51 normal blood donors ( ) and 80 HIV-1 seropositive individuals ( ). divided in three groups according to the Walter Reed Classification (WR I-II, WR III-IV, WR V-VI). Horizontal bars: mean values of each group of subjects. ' HIV-1 subjects, independently of the stage of the disease, and HIV-1 individuals (Table 1). Enriched CD4+ cells showed no detectable level of IL-6 in the absence of a specific stimulus. After addition of 10 µg/ml "' M TPA, CD4+ cells started to release IL-6 in PHA + 10 culture supernatant, although we cannot exclude that the small cells is due to amount of IL-6 produced by enriched CD4+ contaminating monocytes in the cultures. Once again, no statis¬

Several authors have postulated that HIV-1 replication is intrinsically linked to the immune cytokine network in an autocrine mechanism.24-27 In particular, a rapid and transient increase in IL-6 gene expression in monocytes exposed to HIV-1l7 and HIV-1 replication in monocyte cultures accompa¬ nied by a sustained secretion of IL-6'8 have been described. Elevated plasma levels of IL-6 in HIV-1 individuals have also been reported.'8'9 Individuals infected with monocytotropic strains of HIV-1 showed the highest levels of IL-6, in agreement +

with the observation that monocytes but not CD4+ cells can produce high levels of IL-6 in vitro.28'29 Other groups, however, reported that HIV-1 infection as well as the addition of viral components to normal PB light-density mononuclear cells or monocytes do not affect cytokine produc¬ tion.20'21 Moreover, the amount and location ofIL-6-producing cells in HIV-1 lymph nodes were found strikingly similar to those of non HIV-1 hyperplastic lymph nodes.30

Table I. Immunoenzymatic Determination of IL-6 Levels in Culture Supernatants of PB Monocyte and CD4+ T-Lymphocyte Cultures" IL-6

(ng/ml) Normal blood donors

HIV-1 + individuals

Unstimulated monocytes Stimulated11 monocytes

Stimulated' CD4+ T-cells

WRI-1I

III-IV

V-VI

(N°20)

(N°7)

( 3)

(N°35)

1.7 ±0.7 13.2 ± 2.8 1.1 ± 0.3

1.6 ± 1.0 11 ± 3.3 1.5 ±0.3

1.3 ±0.5 11.9 ± 2.5 1.35 ± 0.2

1.45 12.2 1.2

0.4 3.0 0.2

Data are the mean ± SEM of five separate experiments performed in triplicate. No statistically significant differences were observed in IL-6 production between cultures treated with recombi¬ nant HIV env glycoproteins and controls. hMonocyte cultures were stimulated by 1 pg/ml LPS. CCD4+ T-cells were stimulated by 10 µg/ml phytohemagglutinin plus 10'° M phorbol miristate acetate. "

1294

RE ET AL

study aimed to investigate plasma levels of IL-6 an using immunoenzymatic assay in a large series of HIV-1 individuals belonging to different stages of the disease and The present

+

HIV-1 blood donors. Plasma IL-6, detectable only in a subset of HIV-1 + individu¬ als (45 of 80 subjects) and HIV-1 blood donors (28 of 51 subjects), showedatwofoldincrea.se in HIV-1-infected carriers. Although the difference between the two groups was statistically significant (p < 0.01), it was not comparable to the 16-fold or more increase described by others, who used a biological Such discrepancy could be partially due to the assay. different assays used to determine the amount of plasmatic IL-6, but it is interesting to note that IL-6 values in the group of HIV-I1 individuals were similar to the ones reported in other studies, whereas the levels of IL-6 in our HIV-1 controls were much higher. These differences may be explained by the fact that we examined a large number of HIV-l blood donors (51 subjects) than those investigated in other studies. Approxi¬ mately 50% of HIV-1- blood donors in our study showed undetectable levels of plasmatic IL-6, so that the low number of HIV-1 controls (11 to 15) examined in previous reports might not be representative of the physiological status of the normal population. The lower proliferation of IL-6-dependent cell lines in the presence of normal plasma could also be due to some inhibitory factor(s) lacking in HIV-I+ plasma rather than to a different IL-6 production. It is noteworthy that ARC/AIDS patients showed significantly higher levels of IL-6 not only with respect to HIV-1- blood donors, but also to HIV-1+ asymptomatic individuals, whereas IgG levels were similar in all HIV-1-infected subjects. In + particular, in HIV-1 asymptomatic subjects (WRI-II), IL-6 plasma levels were not different from HIV-1 blood donors, whereas IgG levels were significantly already elevated. Al¬ though ARC/AIDS patients were free of intercurrent illness at the time of the study, subacute bacterial and viral infections could explain the higher levels of plasmatic IL-6 in advanced stages of the disease. Since the group of HIV-1+ individuals with detectable levels of plasma IL-6 showed increased levels of IgG with respect to + HIV-1 individuals with undetectable levels of plasma IL-6, we cannot exclude that IL-6 play some role in inducing the hyper¬ gammaglobulinemia frequently associated with HIV-1 infec¬ tion. However, hypergammaglobulinemia may depend on chronic stimulation by HIV-1 antigens or chronic infection with HIV-I in vivo, triggering the production of specific anti-HIV antibodies. Moreover, the HIV-1-induced loss of T-cell-mediated immunoregulatory mechanisms that control cells infected with other lymphotropic viruses, such as Epstein-Barr virus and cytomegalovirus may also contribute to the hypergammaglobu¬ linemia associated with HIV-1 infection.31 Since an increased spontaneous production of IL-6 by PB cells has been reported,19 in a further group of experiments we analyzed IL-6 production by purified monocytes and CD4+ lymphocytes. We were unable, in our experimental conditions, to detect any significant difference in the amount of IL-6 released in monocyte and CD4+ T-cell culture supernatants between HIV-1+ individuals and HIV-1 blood donors, as well + as among HIV-1 subjects belonging to different stages of the disease. -

"

-

In conclusion, our data demonstrate that HIV-1 + individuals showed a twofold increase in the plasmatic levels of IL-6 with respect to HIV-1 controls, which may contribute to explain the hypergammaglobulinemia constantly observed in HIV-1 infec¬ tion. Nevertheless, the lack of a clear correlation between plasma levels of IL-6 and gammaglobulins do not support the hypothesis that an increased production of IL-6 is the main factor involved in the pathogenesis of HIV-1 -associated hypergamma¬ "

globulinemia.

ACKNOWLEDGMENTS This research

was

Ministry of Health,

supported by Progetto AIDS of the Italian by CNR target project Fat.Ma.

Rome and

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Address reprint requests to: Michele La Placa. M.D. Institute of Microbiology

Prof.

University of Bologna Via Massarenti 9 40138 Bologna, Italy