RANTES in onchocerciasis: changes with ivermectin treatment

RANTES in onchocerciasis: changes with ivermectin treatment

P. J. COOPER*, R. H. GUDERIAN*, D. PRAKASHy, D. G. REMICKy, I. ESPINEL*, T. B. NUTMANz, D. W.TAYLORx & G. E. GRIFFIN Division of Infectious Diseases, St George’s Hospital Medical School, London, UK,

*Onchocerciasis Control Programme, Hospital Vozandes, Quito, Ecuador,yDepartment of Pathology, the University of MichiganMedical School, MI andzLaboratory of Parasitic Diseases, National Institutes of Health, MD, USA, andxCentre for Tropical

Veterinary Medicine, University of Edinburgh, Edinburgh, UK

(Accepted for publication 31 July 1996)

SUMMARY

Adverse reactions are seen relatively frequently after treatment of onchocerciasis patients withivermectin. The chemokines RANTES and IL-8, which have both chemotactic and activation propertiesfor eosinophils and neutrophils, respectively, may have a role in the pathogenesis of post-treatmentreactions. Circulating levels of the chemokines and the cytokines tumour necrosis factor-alpha (TNF-®)and IL-6 were measured in the plasma of 22Onchocerca volvulus-infected subjects. Peaks ofmean circulating levels of RANTES and TNF-® were seen at 6 h after ivermectin administration.Peripheral eosinophil counts declined at 36 h post-treatment and an early peak in RANTES levels wasassociated with a delay in peripheral eosinopenia. RANTES levels were negatively correlated withseverity of rash (P< 0.001) and lymphoedema (P< 0.05), suggesting that high circulating levels ofRANTES may inhibit eosinophil sequestration. No changes in circulating levels of IL-8 were seen.These findings suggest a possible role of circulating RANTES in modulating eosinophil sequestrationin vivo.

Keywords onchocerciasis ivermectin chemokines RANTES cytokines

INTRODUCTION

Onchocerciasis is caused by infection with the filarial parasite,Onchocerca volvulus, which is transmitted by the bite of blackfliesof the genusSimulium. An estimated 18 million people are infectedworldwide in equatorial Africa, the Yemen, and Central and SouthAmerica [1]. The infection causes debilitating dermal and lym-phatic pathology and is a leading infectious cause of visual loss andblindness.

Treatment of onchocerciasis patients with microfilarici-dal drugs is associated with transient adverse reactions (theMazzotti reaction) characterized by pruritus, rash, fever, adenitis,arthralgia, and postural hypotension. Severe and life-threateningreactions were commonly seen after treatment with diethylcarba-mazine (DEC) [2,3], which has now been replaced by ivermectin, ahighly effective microfilaricide associated with less severe andless frequent adverse reactions [4]. Nevertheless, between 10%and 30% of ivermectin-treated patients with onchocerciasismay experience post-treatment reactions [5–8] which may besevere [9].

The pathophysiology of the post-treatment reaction is poorlyunderstood. The severity of the reaction appears to be related toinfection intensity [5,10], and the close relationship betweenmicrofilarial killing and the appearance of eosinophils and theirsecretions in the skin and lymph nodes after treatment withmicrofilaricidal drugs has suggested a central role for eosinophilsin this reaction [11–14]. Neutrophils, which, like eosinophils, areable to kill O. volvulusmicrofilariaein vitro [15,16], may also beinvolved, and high circulating levels of elastase, a marker ofneutrophil granule release, have been reported following ivermec-tin therapy [17]. Evidence has also emerged for a role of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-�) andIL-6 in the pathogenesis of this reaction [18,19].

The chemokines RANTES (regulated on activation normal Tcell expressed and secreted) and IL-8 are thought to have a role inthe selective recruitment of eosinophils and neutro-phils, respectively, to sites of inflammation. RANTES has chemo-tactic properties for monocytes, memory CD4+ T lymphocytes andeosinophils [20–22], while IL-8 is able to activate and chemoat-tract neutrophils [23]. A role for RANTES has been proposed inallergic disorders [24] which share many of the immunologicalfeatures seen in onchocerciasis [25]. Because chemokines mighthave a role in the pathogenesis of the post-treatment reactions in

Clin Exp Immunol 1996;106:462–467

462 # 1996 Blackwell Science

Correspondence: Philip Cooper, Laboratory of Parasitic Diseases,Building 4 Room 126, National Institutes of Health, Bethesda,MD 20892, USA.

onchocerciasis, particularly in the recruitment and activation ofimmune effector cells and granulocytes from the peripheral circu-lation to sites of inflammation in the tissues, we measured levels ofRANTES, IL-8, and other pro-inflammatory cytokines in theplasma of O. volvulus-infected patients for 7 days followingivermectin administration.

MATERIALS AND METHODS

Study populationThe study was performed in anO. volvulus-hyperendemic area inEsmeraldas Province in Ecuador [26]. Twenty-two inhabitantswere recruited from the community of Tigre along the RiverCayapas. None of these subjects had previously received antifilar-ial chemotherapy. Children less than 5 years old and pregnantwomen were excluded from the study. Informed verbal consentwas obtained from all study participants.

Treatment protocol and sample collectionIvermectin treatment. All subjects received a single standard

treatment dose of ivermectin orally (150�g/kg) after beingweighed. Administration of the drug and the treatment and obser-vation of subsequent reactions were performed by a medical teamwhich remained in the community for 72 h after treatment andreturned at 7 days for follow up.

Blood and tissue samples. Samples were taken at nine timepoints: 0, 6, 12, 24, 36, 48, 60, 72 and 168 h (7 days). The followingsamples were taken at each time point: skin snips from both iliaccrests using a Stolz corneoscleral punch, thick and thin blood films,and 7 ml of venous blood. Blood was drawn from the antecubitalfossa using a vacutainer containing EDTA. Blood samples werecentrifuged immediately and the plasma was stored in endotoxin-free cryotubes in liquid nitrogen.

Skin biopsies were placed in 96-well plates containing physio-logical saline and left for 6 h at ambient temperature beforecounting the number of emergent microfilariae using an invertedmicroscope. After counting, a drop of 0.2M EDTA was added toeach well (final concentration 0.1M EDTA) and the plates werestored at 48C for further analysis. The snips were blotted dry,weighed, and resuspended in 0.1M EDTA. A highly sensitive andspecific polymerase chain reaction (PCR) assay for the detection ofO. volvulusin skin snips was used, as previously described [27].

Thick and thin blood films were stained with Giemsausing standard methods. The thick films were examined for thepresence of malarial parasites and thin films for estima-tion of differential leucocyte counts. Leucocyte counts wereperformed using the Unopette microcollection system (BectonDickinson, Rutherford, NJ) according to the manufacturer’sinstructions.

Grading of adverse reactionsEach patient was examined at baseline and at each of thesampling time points. A specially designed chart was used whichnoted and graded the presence of the following: temperature,pulse rate, standing and lying blood pressure, lymphadenopathy,lymphoedema, headache, myalgia, athralgia, and ocular com-plaints. The same two observers performed all clinical assess-ments. Reactions were graded using a modified scoring systembased on that used by Awadzi [28]. A score was calculated for eachtime point and each patient’s overall reaction score was taken asthe highest score at any time point after treatment (e.g. 6–168 h).

The background score (e.g. score at time 0) was subtracted fromthe peak score.

Serum cytokine and chemokine measurementsTNF-� activity. TNF bioactivity was assayed as previously

described [29]. Briefly, a highly sensitive cell line, WEHI subclone13, was used which detects TNF at a concentration of 2 pg/ml [30].Samples were serially diluted in 96-well microtitre plates and theWEHI resuspended at 5�104 cells/ml in RPMI 1640 with 10%fetal calf serum (FCS), 2 mM L-glutamine, 30�g/ml gentamicin and0.5�g/ml actinomycin D and incubated at 378C in a humidifiedatmosphere containing 5% CO2 for �20 h. The following day, celllysis was detected by adding MTT-tetrazolium (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolblue) and incubating the plates for a further 4 h. The supernatantwas discarded and the dark blue MTT crystals were dissolved in0.04N HCl/isopropanol. After an overnight incubation at roomtemperature in the dark, absorbance of the plates was measuredusing a microplate reader (Bio-Tek Instruments, Winooski, VT).Units of TNF were calculated using recombinant standards (CetusCorp., Emeryville, CA).

IL-6 assay.Plasma IL-6 levels were measured using a B-9 cellline which proliferates in response to IL-6 [31]. Briefly, serialdilutions of the plasma samples were placed in 96-well microtitreplates, to which were added 5�104 B-9 cells in Iscove’s modifiedDulbecco’s medium (GIBCO, Grand Island, NY) containing5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100�g/ml strep-tomycin, and 100 mM 2-mercaptoethanol. Cells were incubated for72 h at 378C, and pulsed for the final 6 h with MTT-tetrazolium andprocessed as described above. Recombinant human IL-6 (Pepro-Tech, Rocky Hill, NJ) was used as standard.

IL-8 ELISA.IL-8 was measured as previously described [32].Microtitre plates were coated with anti-IL-8 polyclonal rabbit seraand incubated overnight at 48C. After washing in PBS with 0.05%Tween 20, the plates were blocked with 2% bovine serum albumin(BSA) in PBS for 1 h at 378C. Serum samples were added and theplates incubated at 378C for 1 h. After washing, biotinylated IL-8was added and the plates incubated at 378C for 30 min. Avidin-horseradish peroxidase (Dako Corp., Carpinteria, CA) was addedand the plates were incubated at 378C for 30 min. The plates weredeveloped using substrate solution (0.67 mg/ml orthophenylene-diamine chloride (Dako) with H2O2 in 25 mM citrate phosphatebuffer pH 5.0). Standard curves were calculated using recombinanthuman IL-8 standards (PeproTech).

RANTES ELISA.RANTES was measured by ELISA using acommercial kit (R&D Systems, Minneapolis, MN) following themanufacturer’s instructions.

Statistical analysisSkin microfilarial intensities are expressed as the geometric meannumber of microfilariae per milligram of skin (mf/mg). Cytokinelevels are expressed as either geometric means or as a percentagechange from the baseline measurement (at 0 h). The associationbetween two continuous variables was calculated using Spear-man’s rank correlation coefficients. The comparison of two meanswas calculated using a pairedt-test and of more than two meansusing two-way analysis of variance. Multiple comparisons of asingle variable were tested using a modifiedt-test with theBonferroni adjustment. Statistical significance is inferred byP< 0.05.

RANTES and onchocerciasis 463

# 1996 Blackwell Science Ltd,Clinical and Experimental Immunology, 106:462–467

RESULTS

Skin microfilarial intensity and haematological indicesThe pretreatment geometric mean microfilarial infection intensitywas 7.5 mf/mg (range 0–150 mf/mg). Of the 22 patients studied, sixhad no detectable dermal microfilariae by microscopy, though atthe second time point (6 h) two of these had become positive.However, all subjects were positive by PCR on at least one timepoint following treatment. By 7 days post-treatment the micro-filarial intensity had dropped to 0.3 mf/mg skin, but six remainedskin snip-positive. Microfilarial levels started to decline at 24 hafter treatment (Fig. 1), but this decline was not statisticallysignificant until 36 h post-treatment. Eosinophil counts were ele-vated at time 0 (mean 1012 cells/ml, 95% CI 771–1329), rosesignificantly at 6 h after treatment (P < 0.05), started to fall at 24 hat the same time as skin microfilarial levels started to decline(Fig. 1), and reached a nadir at 36 h (mean 712, 95% CI 600–844)(comparison witht� 0; P< 0.05). Thereafter, eosinophil levelsrose again to a peak at 60 h (mean 1880, 95% CI 1480–2387)(comparison witht�0; P< 0.01) and declined to below pretreat-ment levels by day 7 (mean 453, 95% CI 306–670) (comparisonwith t�0; P< 0.01). Neutrophil levels were 3939 cells/ml (95% CI3475–4465) at time 0 and rose gradually over the study period to amean of 5167 cells/ml (95% CI 4370–5167) at 7 days, which wassignificantly higher than baseline (P< 0.01).

Adverse reactions to ivermectinA high frequency of adverse reactions was seen. One patient had asevere reaction characterized by fever (398C) and systemic pos-tural hypotension at 24 h post-treatment. Most reactions were seenafter 36 h and were characterized by fever (7/22 or 32%), urticarialor maculopapular rashes (9/22 or 41%), tender regional lympha-denopathy (7/22 or 32%), and lymphoedema (8/22 or 36%). Othercomplaints included headache, myalgia, athralgia, lacrimation,photophobia, and orchitis. The mean reaction score peaked at60 h (Fig. 1).

Chemokine profilesHigh plasma levels of RANTES were seen after ivermectintreatment. At the time of treatment (t� 0 h), mean levels

of RANTES were 13.0 ng/ml (95% CI 10.1–16.6). A peak ofcirculating RANTES was seen 6 h post-treatment (32.7 ng/ml,95% CI 27.4–39.0), which was significantly greater than baselinelevels (P< 0.001). RANTES levels remained high andonly returned to baseline levels at 72 h post-treatment (mean13.0 ng/ml, 95% CI 11.3–14.9). The percentage change comparedwith baseline of RANTES levels after treatment with ivermectinare shown in Fig. 2a. Mean baseline levels of IL-8 were 55 pg/ml(95% CI 23–133). IL-8 levels peaked at 12 h (Fig. 2b), but therewere no significant differences between time points.

Cytokine profilesMean pretreatment levels of TNF-� were 57 pg/ml (95% CI 49–65). Levels of TNF-� were significantly elevated at 6 h (150 pg/ml; 95% CI 127–178) (P< 0.001) and declined rapidly to pre-treatment levels by 24 h. When expressed as a percentage ofbaseline levels, a similar relationship was obtained (Fig. 2c).Mean IL-6 levels were 45 pg/ml at baseline (95% CI 25–81) andpeaked at 60 h (Fig. 2d), though there were no statistical differ-ences between time-point values.

Relationships between adverse reaction scores, infection intensity,and cytokine and chemokine levelsPeak reaction score was significantly associated with microfilarialintensity at time 0 (P< 0.01). Peak IL-6 levels (at 60 h) weresignificantly associated with peak reaction score (P< 0.05), thepresence of fever (P< 0.05), and baseline microfilarial counts(P< 0.001). Peak RANTES levels were significantly nega-tively correlated with severity of rash (P< 0.001) and lymphoedema(P< 0.05). The time to peak of RANTES levels was negativelycorrelated with time to trough of eosinophil levels (P< 0.01) (e.g.early peak RANTES levels were associated with delayed peripheraleosinopenia)

DISCUSSION

Eosinophils are thought to be of central importance in the patho-genesis of post-treatment reactions in onchocerciasis [11,13]. Thisreaction is characterized by the development of an eosinophil-richinfiltrate at the sites of inflammation in the skin and lymph nodeswith the release of large quantities of eosinophil-derived peptides[11–14]. Recently, there has been much interest concerning therole of RANTES in the selective recruitment of eosinophils inallergic inflammation [33]. The effects of RANTESin vitroinclude the activation of eosinophils [34], eosinophil exocytosis[21,24,34], and an increase in the expression of adhesion moleculeswhich are important in transendothelial migration (TEM)[22,33,34]. The intradermal injection of recombinant humanRANTES into dogs resulted in the development of an inflamma-tory infiltrate composed predominantly of eosinophils and mono-cytes [35]. High expression of RANTES is seen at the sites ofinflammation in patients with allergy [36,37].

RANTES-induced TEM requires a concentration gradient ofthe moleculein vitro [22]. There is evidence that free IL-8 in thecirculation may actually inhibit neutrophil migration [38,39]. Thismay also be true for the eosinophil chemotactic effect of RANTES.In support of this was the finding that an early peak in RANTESlevels was associated with a delay in the fall in peripheraleosinophil counts. The peripheral eosinopenia was seen coincidentwith the decline in dermal microfilarial levels (Fig. 1). This has

464 P. J. Cooperet al.


Fig. 1. Relationship between skin microfilarial intensity (mf/mg),blood eosinophil counts (cells/ml), and reaction score followingtreatment with ivermectin in 22 subjects.&, Microfilarial intensities;*, eosinophil counts; the shaded area represents the area under the reactionscore graph.

also been described following DEC administration [10,13], atwhich time the number of eosinophils in the skin increases andeosinophil-mediated microfilarial killing is seen [11,13]. Negativecorrelations were seen between severity of rash and lymphoedemaand circulating levels of RANTES. The rash associated withadverse reactions following treatment with DEC is associatedhistologically with eosinophil infiltration and degranulationaround dead and degenerating microfilariae [11,13]. The lymphoe-dema may be due to the local effects of eosinophil degranulation[40] and/or lymph stasis secondary to the inflammatory reactionsassociated with large numbers of dead and degenerating micro-filariae in the regional lymph nodes [14]. It is important todifferentiate between the possible effects of high circulatinglevels of RANTES in the peripheral circulation and local produc-tion in the tissues: the findings of this study suggest that the formermay inhibit TEM and modify clinical reactions by abolishing aconcentration gradient, while the latter is known to stimulateeosinophil TEM [22,35].

The high circulating levels of RANTES might have beeninduced by TNF-� [41–43], the levels of which peaked earlyalso. Numerous cell types are capable of secreting RANTES,including platelets, epithelial cells, fibroblasts, macrophages, andendothelial cells [41,44,45].

Typical adverse reactions were seen in a high proportionof patients. A number of large studies have reported similarfindings [5,6,8,9,46]. The kinetics of this reaction differed fromthose described for DEC, which may occur as soon as 1 h aftertreatment [3]. In this study, significant adverse clinical reactionswere not seen until 24 h after treatment (Fig. 1), at which timedermal microfilarial levels started to decline. Such a sequenceof clinical events has also been described by a number of otherinvestigators [8,47]. The differences in timing of reactions follow-ing the two drugs clearly reflects the timing of microfilaricidalkilling. With DEC, evidence of microfilarial killing is seen within5 h of treatment [11,13], while with ivermectin, evidence of killingis not seen before 24 h [48]. Histologically, eosinophils seem to bethe primary effector cell in microfilarial death, and the presence oflarge quantities of secreted eosinophil products (myelin basicprotein (MBP), eosinophil cationic protein (ECP), eosinophilperoxidase (EPO), and Charcoat–Leyden crystal (CLC) protein)are seen in the vicinity of dead and degenerate microfilariae[11,13,14].

A relationship was seen between pretreatment microfilarialintensity and severity of reaction. A similar relationship has beendescribed in DEC-treated patients [10,18] and suggests that micro-filariae or their degradation products play a role in post-treatmentreactions. Previous studies have demonstrated that levels of TNF-�

[18] and IL-6 [18,19] were elevated following microfilaricidal

therapy inO. volvulus-infected subjects but remained unchangedin endemic controls without evidence of infection. No differencewas seen in levels of any of the cytokines and chemokines assayedand infection status (data not shown), which is explained by thefinding ofO. volvulusDNA in skin biopsies taken from all subjects.Peak IL-6 levels correlated with both pretreatment intensity ofinfection and the occurrence and severity of clinical symptomsafter treatment. Such a relationship has been described by others[18].

The mechanisms underlying the adverse reactions whichfollow ivermectin treatment are of interest, as the pathophysio-logical mechanisms are likely to be similar to those occurringin chronic disease [12], and as adverse reactions reduce compli-ance to treatment, an understanding of the mechanisms may lead tothe development of interventions that may be important in achiev-ing long-term disease control. RANTES levels were negativelycorrelated with severity of rash and lymphoedema, which suggeststhat high circulating levels of RANTES may actually inhibiteosinophil sequestration and suppress local adverse effects. Furtherstudies are required to investigate the relationship between med-iators of the systemic and local immune responses in post-treat-ment reactions.

ACKNOWLEDGMENTS

This study was funded by the Wellcome Trust. The inhabitants ofthe community of Tigre are gratefully acknowledged for their co-operation.The following are also thanked for their help in carrying out thisstudy: Raquel Lovato, Martin Dedicoat, Alfonso Anãpa, GregorioMontano, Tom Chiller, David Gaus, Helen Oakey, Wilson Paredes andManuel Calvopinã.

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RANTES in onchocerciasis: changes with ivermectin treatment