Immune Dysregulation to Ethiopian Immigrants in Israel: Relevance to Helminth Infections?

Clin Exp Immunol 1996; 103:239–243

Immune dysregulation to Ethiopian immigrants in Israel: relevance to helminth infections?

Z. BENTWICH, Z. WEISMAN, C. MOROZ*, S. BAR-YEHUDA & A. KALINKOVICH R. Ben-Ari Institute of Clinical Immunology, Kaplan Hospital, Hebrew University Hadassah Medical School, Rehovot, and *Felsenstein Medical Research Centre, Beilinson Medical Campus, Sackler Faculty of Medicine, Tel Aviv University, Petach-Tikva, Israel

(Accepted for publication 3 October 1995)

SUMMARY The infectious disease background and particularly the helminth infections that are endemic in Africa

could have profound effects on the host immune system. Studies that we have performed on an Ethiopian HIVÿ immigrant population that has recently reached Israel, lend support to this notion. They have indeed revealed a very high prevalence of helminth and several other infections with an extreme immune dysregulation, consisting of: (i) highly elevated plasma IgE, IgG, placental isoferritin, p75 soluble TNF receptor (sTNFR) levels and very high blood eosinophilia; (ii) increased secretion from

phytohaemagglutinin (PHA)-simulated peripheral blood mononuclear cells (PBMC) of the cytokines IL-2, IL-4, IL-10 and p75s sTNFR, and decreased secretion of interferon-gamma (IFN-) and IL-6; (iii) increased and decreased surface expression of p75 TNFR and IL-6 receptor on lymphocytes, respectively. The causal relationship between this immune dysregulation and the infectious background is highly suggestive, and could have far-reaching implications in the resistance to other infections.

Keywords cytokines immune dysregulation T helper cell subsets helminth infections Ethiopian immigrants

INTRODUCTION independently of the HIV infection status. These findings have prompted this study. One of the prominent features of the health situation in Africa and other developing countries is the high endemic prevalence of chronic infectious diseases [1]. The host confronted with the SUBJECTS AND METHODS infectious burden would be expected to mount prolonged immune response to this challenge which would alter the Human subjects normal immune balance. Such changes could also have profound Two groups of clinically healthy HIVÿ individuals were studied: effects on the ability to cope with other infections and could also (i) Ethiopian immigrants, shortly after their arrival in Israel; and affect the general and specific immune response to other stimuli. (ii) non-Ethiopian Israelis, matched for age and sex. A striking characteristic of the AIDS epidemic in Africa is the different pattern of the disease with its mode of transmission, rapid Immunoglobulin and placental isoferritin levels spread and fast progression [2–7]. The reasons for this different Plasma IgA and IgE were determined by routine nephelometry pattern are not clear. We have recently suggested that changes in (Beckman IgG; Beckman Instruments, Inc., Galway, Ireland) and the host immune response caused by endemic infections and fluorimetry (Delfia Total IgE Kit; Wallac Oy, Turku, Finland) mostly helminth infections could account for at least a part of techniques. Serum placental isoferritin (PLF) was determined by this pattern [8]. ELISA as described [9]. Briefly, CM-G-8 MoAb was coupled to a This idea has been supported by our experience and studies of 96-well Maxisorp Nunc Immunoplates (Nunc, Roskilde, Den- an Ethiopian immigrant population that has recently mark), and test sera were added to the wells. After incubation reached Israel. They have indeed revealed a very high prevalence and washing, alkaline phosphatase (Sigma, St Louis, MO)-con- of many infections (helminth infections being almost universal), jugated CM-h-9 MoAb was added to each well. Colour was as well as a high prevalence of HIV-1. Furthermore, an extreme developed by the addition of p-nitrophenyl-phosphate substrate degree of immune dysregulation was observed in this population, (Sigma), and read in an ELISA reader at 405 nm. The amount of PLF that bound 250 pg of alkaline phosphatase-conjugated CM-h-9 Correspondence: Professor Zvi Bentwich, R. Ben-Ari Institute of MoAb was arbitrarily considered to be 10 U of PLF, and the

Clinical Immunology, Kaplan Hospital, 76100 Rehovot, Israel. results were expressed as U/ml. 5 1996 Blackwell Science 239 240 Z. Bentwich et al.

Cell culture supernatant preparation absorbed to 96-well Maxisorp Nunc Immunoplates. After 2 h Peripheral blood mononuclear cells (PBMC) were obtained from incubation at 37VC, the wells were blocked with 1% bovine heparinized venous blood by standard centrifugation over Lym- serum albumin (BSA) for an additional 2 h, washed and rabbit phoprep (Nycomed Pharma As., Oslo, Norway), washed and polyclonal antisera to the sIL-6R or to the p75 sTNFR were added

V resuspended at 2 10 /ml in RPMI 1640 (Biological Industries to the wells. After overnight incubation at 4 C the plates were Co, Beit-Haemek, Israel) supplemented with 5% heat-inactivated washed and incubated for 2 h with horseradish peroxidase-con- human AB serum (Sigma), 2 mML-glutamine and antibiotics— jugated puriﬁed goat anti-rabbit IgG (Jackson Labs, West Grove, penicillin, streptomycin and nystatin (Biological Industries). Cells PA). The assay was developed using ABTS (Sigma) as a substrate. (1 ml/well) were cultured in 24-well Multidish plates (Nunc) at The enzymatic product was determined colorimetrically at 405 nm.

. 37VC under 6 5% CO2 with and without phytohaemagglutinin Puriﬁed sIL-6R [14] and p75 sTNFR [17] served as standards. The (PHA; 1:100) (Difco PHA-P; Detroit, MI) for 72 h. For IL-2 detection limit of both assays was 50 pg/ml. The results are

5 production, PBMC (1 10 /well) were seeded in 96-well ﬂat- expressed as ng/ml. All components used for sIL-6R and p75 bottomed plates (Nunc) in RPMI 1640 medium for 1 h at 37VC, sTNFR determination were kindly provided by D. Novick and D. . 6 5% CO2. Subsequently, PHA (1:100) and human anti-IL-2 Wallach, respectively (The Weizmann Institute of Science, Israel).

. receptor antibody, anti-Tac (2 5 "g/ml) (kindly provided by Hoffman-La Roche Inc, Nutley, NJ) (to prevent IL-2 consump- Cytokine membrane expression

" V tion by the stimulated cells), were added and the cells were PBMC (1 10 ,50 l) were incubated at 4 C for 30 min in PBS

. . cultured for 6 days at 37VC, 6 5% CO2 [10]. All supernatants supplemented with 2% heat-inactivated FCS, and 0 1% NaN3 with

. V " (SN) were collected by centrifugation and stored at ÿ70 C 10 g/ml of 48 8 anti-IL-6R [14] or anti-p75 TNFR [16] MoAbs, or until tested. with matched MoAbs (Becton Dickinson, San Jose, CA) served as

a background isotype negative control. After staining, cells were Cytokine and cytokine receptor determination washed twice and then incubated at 4VC for 30 min with FITC-

IL-2 activity in SN was assessed as the ability to stimulate the conjugated goat anti-mouse F(abH)2 fragment of afﬁnity-puriﬁed proliferation of the IL-2-dependent mouse cell line, CTLL, as antibodies to Fab fragment of mouse IgG (BioMakor, Rehovot,

4 described [10]. Briefly, 1 10 CTLL/well and three-fold suc- Israel). Cells were washed and analysed by flow cytometry with a cessive dilutions of SN were cultured for 24 h, pulsed with 1 "Ci of FACScan (Becton Dickinson). The percentage of membrane fluor- 3H-thymidine (Rotem Industries Ltd, Nuclear Research Center- escent cells were acquired on a log scale (5000 events per sample) Negev, Israel) and harvested 18 h later. Results are expressed as on gated lymphocytes defined in forward versus side scattering. mean ct/min for three replicate wells for a given SN dilution. For determination of IL-6 activity in plasma, B9 cells were Statistical analysis used in a standard assay as described [11]. Briefly, serial dilutions Comparisons between groups were performed by the Wilcoxon of heat-inactivated plasma samples and recombinant Escherichia two-sample test, using the Epistat software (Epistat Services, coli-derived IL-6 (Amersham, Aylesbury, UK) were distributed to Richardson, TX). 96-well flat-bottomed plates (Nunc). Five thousand B9 cells in 0.1 ml culture medium consisting of RPMI 1640, 10% fetal calf RESULTS serum (FCS; Biological Industries), 2 mML-glutamine and antibiotics, were added into each well. Plates were incubated for 72 h at Immune activation markers in the blood

. 3 37VC under 6 5% CO2 and pulsed with H-thymidine for the last Several immune activation markers were determined in the blood 18 h. Data are expressed as U/ml calculated from the standard of the studied population and are depicted in Fig. 1. As can be seen, curve of rIL-6. plasma levels of IgG, IgE, and serum levels of PLF, were

For determination of IL-6 activity in SN, 35 000 T-1165 cells signiﬁcantly higher in the Ethiopian population in comparison

X . [12] were cultured with serial dilutions of the SN and human rIL-6 with the non-Ethiopian control group (P ` 0 001–0 01). Most

4 (15 10 reference units/mg protein) (InterPharm, Ares-Serono strikingly, the mean plasma levels of IgE among the Ethiopian Group, Nes-Ziona, Israel) in 96-well ﬂat-bottomed plates (Nunc), immigrants reached values of over ﬁve-fold more than mean levels

. X X X in 0 1 ml of RPMI 1640 culture medium containing 10% FCS, among the non-Ethiopian controls, the means s e m being

5 X X X X

4mM L-glutamine, 2 10 M 2-mercaptoethanol and antibiotics. 753 8 123 6 U/ml and 143 6 33 6 U/ml, respectively. Interest-

. After an incubation period of 48 h at 37VCin65% CO2, the wells ingly, it should also be noted that such extreme elevations of

were exposed to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- plasma IgE among the Ethiopians were not accompanied by any lium bromide (MTT; Sigma) for 2 h at 37VC as described [13]. allergic manifestations. The difference in serum levels of PLF Subsequently, 0.1 ml solution of 20% SDS (Sigma) 1:1 water/N,N between the groups was even more pronounced, the

. X X X dimethylformamide (Sigma) pH 4 7, was added overnight. Absorp- mean s e m being 10-fold more in the Ethiopians compared

tion was measured at 450 nm and the data were calculated from the with non-Ethiopians, 52 38 U/ml and 5 9 U/ml, respectively.

standard curve of human rIL-6. Blood eosinophil counts were above the normal range in half of the Interferon-gamma (IFN-), IL-4 and IL-10 levels in SN were Ethiopian studied population (not shown). Cytokine studies

determined by ELISA using Cytoscreen Immunoassay Kits (Bio- revealed signiﬁcantly elevated levels of plasma p75 sTNFR

X X X

X X X Source Int., Camarillo, CA) according to the manufacturer’s (P ` 0 01), the mean s e m being 4 6 0 3 ng/ml and

X X instructions. 3 5 0 2 ng/ml in the two groups, respectively, while the differ- Plasma levels of sIL-6R and soluble p75 tumour necrosis factor ences in plasma levels of IL-6 and of sIL-6R were not signiﬁcant.

receptor (sTNFR) as well as p75 sTNFR levels in SN were Plasma levels of several other cytokines—Il-2, IL-4, IL-10, and determined by speciﬁc ELISA according to reported procedures IFN-—were found to be undetectable or unreliable, and thus these

[14,15]. Brieﬂy, MoAbs to sIL-6R [14] or to p75 sTNFR [16] were cytokines were measured by their secretion into SN of PBMC. 5 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 103:239–243 Immune dysregulation in Ethiopians 241

Fig. 1. Serum levels of placental isoferritin (PLF) (a) and plasma levels of IgG (b), IgE (c), p75 sTNFR (d), IL-6 (e) and sIL-6R (f) in non-

k Ethiopian Israelis (l) and Ethiopian immigrants ( ). Each point represents a single individual. The horizontal bars indicate arithmetic means. NS, Not signiﬁcant.

Cytokine secretion by PBMC was also clearly elevated in the Ethiopians compared with non-

X X

X X X Secretion of several cytokines by PBMC was elevated in PBMC Ethiopians, the mean s e m being 1 4 0 4 ng/ml compared

X X obtained from Ethiopians in comparison with non-Ethiopian con- with 0 25 0 2 ng/ml, respectively. trols. The results of these studies are depicted in Fig. 2. Of the Th2

type cytokines, secretion of IL-4 and IL-10 was signiﬁcantly Cytokine membrane expression

elevated, 231 64 pg/ml versus 64 13 1 pg/ml and 2115 Expression of cytokine receptors on lymphocyte membranes was

286 pg/ml versus 1141 127 pg/ml (mean s.e.m.), respectively, studied for IL-6R and for TNFR. The two receptors were found to

while that of IL-6 was decreased in the Ethiopians, 1143 182 U/ behave differently, the percentage of lymphocytes expressing p75 ml versus 2399 372 U/ml. TNFR being increased while that of lymphocytes expressing IL-6R

Of the Th1 type of cytokines, secretion of IL-2 was elevated in was decreased, in Ethiopians compared with non-Ethiopians:

. . X . ` the Ethiopians compared with non-Ethiopians, 55 169 931 ct/ 20 8% versus 10 7% (P 0 01), and 24 7% versus 36%

` min, 30 720 598 ct/min, respectively, while the secretion of IFN- (P 0 01), respectively (Fig. 3). The increased expression of

was signiﬁcantly decreased in the Ethiopians compared with p75 TNFR was strongly correlated to the increased secretion of

non-Ethiopians, being 2136 106 pg/ml versus 3706 166 pg/ml, this molecule by PBMC (Fig. 2). The decreased expression of IL- respectively. 6R is probably related to the decreased secretion of the IL-6 (Fig. Secretion of p75 sTNFR, representing the TNF system and 2), though whether this is a causal relationship remains to be

therefore not clearly deﬁned in terms of Th1 or Th2 type cytokines, determined.

Fig. 2. Levels of IL-2 (a), IFN- (b), p75 sTNFR (c), IL-4 (d), IL-10 (e), and IL-6 (f) in supernatants (SN) of peripheral blood mononuclear

h cells (PBMC) obtained from non-Ethiopian Israelis (d) and from Ethiopian immigrants ( ). Cells were cultured for 72 h in the presence of

phytohaemagglutinin (PHA). For IL-2 determination cells were cultured for 6 days. Cytokine levels were determined by bioassays or ELISA

X X X (for details see Subjects and Methods). Data are expressed as mean s e m n, Number of individuals. 5 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 103:239–243 242 Z. Bentwich et al.

Table 1. Rates of some infections in Ethiopian immigrants (%)*

Ascaris lumbricoides 99 Necator americanus 90 Schistosoma mansoni 78 Giardia lamblia 38 Entamoeba histolytica 19 Plasmodium vivax 10 Hepatitis B virus carriers 12 Hepatitis B virus-exposed 78 Treponema pallidum 31 HIV-1 2

Fig. 3. Surface p75 TNFR and IL-6R expression on lymphocytes. Periph- * Data (in round ﬁgures) are from eral blood mononuclear cells (PBMC) obtained from non-Ethiopian Israelis [19] and from our own unpublished

observations. h (d) and from Ethiopian immigrants ( ) were incubated with anti-p75 TNFR or anti-IL-6R MoAb, washed and then incubated with FITC- conjugated goat anti-mouse IgG (for details see Subjects and Methods). inﬂuences could then serve as a major element in shifting the

Cytoﬂuorimetry was performed after gating for the ‘main’ cell population balance toward a Th2 type of immune response.

(containing b95% of lymphocytes) deﬁned in forward versus side scatter- Is the immune activation we have observed in the HIV non-

X X X ing. Data are expressed as mean s e m of the percentage of membrane infected Ethiopian immigrants a Th2 type of activation? The expression. n, Number of individuals. answer to this question is not simple. The Ethiopians had elevated plasma IgE, IgG and eosinophilia, and had increased secretion of IL-4 and IL-10 from their PBMC, all of which would ﬁt with a Th2

DISCUSSION type of response. Furthermore, they also had decreased secretion of

The results of these studies have demonstrated that a broad type of IFN-, indicating a lowered Th1 response. On the other hand, the immune dysregulation is found in HIVÿ new immigrants from decreased secretion of IL-6, and the increased secretion of IL-2 by Ethiopia independent of HIV infection. It consisted of elevated their PBMC, would fit better with a Th1 type of immune response. plasma IgE and IgG, blood eosinophilia and increased serum PLF Thus, our findings clearly indicate a mixed profile of Th1 and Th2,

and p75s sTNFR. Other studies revealed elevated secretion of IL-2, and may reflect the true in vivo state where the prevalence of Th0 IL-4, IL-10, and sTNFR, and decreased secretion of IFN- and IL- cells is more dominant [23,24]. Additional factors that may play a 6 from PHA-stimulated PBMC. Furthermore, expression of cyto- role in our system and can explain the results are: (i) the activated kine receptors on lymphocyte membranes was found to be TNF system, probably as a result of the helminth infections [22], decreased for IL-6R while being increased for p75 TNFR. may affect the cytokine profile [23–25]; (ii) the presence of The presence of such broad immune dysregulation in our study elevated IL-4 does not inhibit the proliferation of Th1 cells group of Ethiopian new immigrants is in all likelihood representa- secreting IL-2 [25]; (iii) the costimulation of memory cells by tive of a general phenomenon in the wide population of Ethiopia other infections is very likely to be continually present, and would and other countries in Africa. It is probably present in other increase at least the IL-2 secretion in vitro by these proliferating developing countries as well, and our recent study of a small cells. population of Thai laborers in Israel that has revealed similar The activation of the PLF has not yet been categorized to one of findings, indeed lends support to this expectation [18]. The most the Th types. As a molecule with a physiological immunosuppres- likely and simple explanation for such wide immune disturbances sive function, it is of great interest that increased levels of this is the very high prevalence of infectious and parasitic diseases molecule are also found during HIV infection and may be corre-

endemic in these areas. This is certainly the case for the Ethiopian lated with the stages of the infection [26]. Activated CD4 T cells new immigrants that we had the oppor-tunity to study upon their are its major source and, thus the extremely high serum levels of

arrival in Israel. As can be seen in Table 1, this population is highly PLF found in the HIVÿ Ethiopian population indicate a high

infested by various types of infections. Note should be taken also of degree of CD4 cell activation, accompanied by a certain degree the relatively high percentage of HIV-1 carriers in this population, of immunosuppression [27]. which is over 100 times the prevalence of HIV-1 infection in the Our findings in the Ethiopian immigrants may be unified under Israeli non-Ethiopian population. the less restrictive general heading of immune activation. Such A central question in the African scenario is whether there is a activation, with the helminth infection in its background, may specific influence for certain infections on the immune profile. One serve to facilitate and influence the course of other infections, of the striking elements in the infectious load in Africa and other including HIV [28–31]. The orchestration of the immune network developing countries are the helminth infections, which are uni- is dependent on a continuous balance among individual cytokines. versal and chronic lifetime infestations [20,21]. As shown above, This accounts for the sometimes polar outcomes of the response such infections were very common among the Ethiopians that we with the same ‘players’. We believe that this also holds true for the studied. Although our understanding of the mechanisms involved in scene in Africa and the developing world, where immune activa- the handling of helminth infections is limited, they result in tion is continuous and the result of chronic persistent stimulation eosinophilia, high levels of IgE and mast cell activation, which [8,32]. The implications of these findings are manifold. On the

are suggestive for increased IL-4 and IL-5 secretion [22]. Such level of public health and preventive measures in Africa and the 5 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 103:239–243 Immune dysregulation in Ethiopians 243 other developing countries, a much greater emphasis on the plasmocytomas for survival and proliferation in vitro. Science 1986; eradication of helminth infections should be given priority. Such 233:566–7. eradication may modulate the baseline immune response and lead 13 Hansen MB, Nielsen SE, Berg K. Re-examination and further devel- to improved ability to cope with infections. On the level of opment of a precise and rapid dye method for measuring cell growth/ protective vaccine development, it is to be expected that the type cell kill. J Immunol Methods 1989; 119:203–10. 14 Novick D, Engelmann H, Revel M, Leitner O, Rubinstein M. Mono- of immune response and success of vaccines would be very clonal antibodies to the soluble human IL-6 receptor: Affinity purifica- different in Africa than in the developed world, and therefore tion, ELISA, and inhibition of ligand binding. Hybridoma 1991; different approaches will have to be used there. 10:137–46. 15 Aderka D, Engelmann H, Hornik V, Skornick Y, Levo Y, Wallach D, Kushtai G. Increased serum levels of soluble receptors for tumor ACKNOWLEDGMENTS necrosis factor in cancer patients. Cancer Res 1991; 51:5602–7. 16 Engelman H, Holtmann H, Brakebusch C et al. Antibodies to a soluble We thank I. Cohen and G. Berke for critically reviewing the manuscript, D. form of tumor necrosis factor (TNF) receptor have TNF-like activity. J Wallach and D. Novick for their generous gifts of the components needed Biol Chem 1990; 265:14497–504. for TNFR and IL-6 determinations, and Hoffman-La Roche Inc., Nutley, NJ 17 Engelman H, Novick D, Wallach D. Two tumor necrosis factor for the generous gift of anti-Tac for the determination of IL-2 secretion. The binding proteins purified from human urine. J Biol Chem 1990; technical assistance of N. Harpaz, E. Porat, L. Horenstein and B. Zingerman is 265:1531–6. gratefully acknowledged. This work was supported by grants from the Israeli 18 Kalinkovich A, Maayan S, Weisman Z, Harpaz N, Bentwich Z. Immune Ministry of Health. activation, a co-factor for HIV transmission in Thailand? Lancet 1994; 343:1506–7. 19 Nahmias J, Greenberg Z, Berger SA, Hornstein L, Bilgury B, Abel B, REFERENCES Kutner S. Health profile of Ethiopian immigrants in Israel: an overview. Isr J Med Sci 1993; 29:338–43. 1 Hori H, Watanabe M, Sakurai M. Infectious diseases in African 20 Hodes RM, Kloos H. Health and medical care in Ethiopia. N Engl J Med children. Acta Pediatr Jpn 1993; 35:553–8. 1988; 319:918–24. 2 Piot P, Goeman J, Laga M. The epidemiology of HIV and AIDS in 21 Bundy DAP, Medley GF. Immuno-epidemiology of human geo- Africa. In: Essex M, Mboup S, Kanki PJ, Kalengayi MR, eds. AIDS in helminthiasis: ecological and immunological determinants of worm Africa. New York: Raven Press, Ltd, 1994: 157–72. burden. Parasitology 1992; 104:5105–19. 3 Anzala OA, Nagelkerke NJD, Bwayo JJ et al. Rapid progression to 22 Maizels RM, Bundy DAP, Selkirk EM, Smith DFS, Anderson RM. disease in African sex workers with human immunodeficiency virus Immunological modulation and evasion by helminth parasites in human type 1 infection. J Infect Dis 1995; 171:686–9. populations. Nature 1993; 365:797–805. 4 The working group on mother-to-child transmission of HIV. Rates of 23 Romagnani S. Lymphokine production by human T cells in disease mother-to-child transmission of HIV-1 in Africa, America and Europe: states. Ann Rev Immunol 1994; 12:227–58. results from 13 perinatal studies. J Acquir Immune Def Syndr Hum 24 Romagnani S. Biology of human Th1 and Th2 cells. J Clin Immunol Retrovir 1995; 3:506–10. 1995; 15:121–9. 5 N’Galy B, Ryder RW, Bila K et al. Human immunodeficiency virus 25 Powrie F, Coffman RL. Cytokine regulation of T-cell function: poten- infection among employees in an African hospital. N Engl J Med 1988; tial for therapeutic intervention. Immunol Today 1993; 14:270–4. 319:1123–7. 26 Moroz C, Misrock SL, Siegal FP. Isoferritins in HIV infection: relation 6 Berkley S. AIDS in the developing world: an epidemiologic overview. to clinical stage, CD8 lymphocyte binding and the pathogenesis of Clin Infect Dis 1993; 17(Suppl.2):S329–36. AIDS. AIDS 1989; 3:11–16.

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