Filarial Nematode Parasites Secrete a Homologue of the Human Cytokine Macrophage

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http://iai.asm.org/cgi/content/full/66/12/5955

 

 

 

Infection and Immunity, December 1998, p. 5955-5963, Vol. 66, No. 12

0019-9567/98/$04.00+0

 

1998, American Society for Microbiology. All rights reserved.

 

Filarial Nematode Parasites Secrete a Homologue of the Human Cytokine

Macrophage Migration Inhibitory Factor

Diana V. Pastrana,1 Nithyakalyani Raghavan,1 Peter FitzGerald,2

Stephen W. Eisinger,1 Christine Metz,3 Richard Bucala,3 Robert P.

Schleimer,4 Carol Bickel,4 and Alan L. Scott1,*

 

Department of Molecular Microbiology and Immunology, School of Hygiene

and Public Health, Johns Hopkins University, Baltimore, Maryland

212051; Division of Computer Research and Technology, National

Institutes of Health, Bethesda, Maryland 208922; Department of

Clinical Immunology, Asthma and Allergy Center, School of Medicine,

Johns Hopkins University, Baltimore, Maryland 212244; and The Picower

Institute for Medical Research, Manhasset, New York 110303

 

Received 7 May 1998/Returned for modification 19 June 1998/Accepted 10

August 1998

 

 

ABSTRACT

 

Filarial nematode parasites establish long-term chronic infections in

the context of an antiparasite immunity that is strongly biased toward

a Th2 response. The mechanisms that lead to this Th2 bias toward

filarial antigens are not clear, but one possibility is that the

parasites produce molecules that have the capacity to proactively

modify their immunological environment. Here we report that filarial

parasites of humans secrete a homologue of the human proinflammatory

cytokine macrophage migration inhibitory factor (MIF) that has the

capability of modifying the activity of human monocytes/macrophages. A

cDNA clone isolated from a Brugia malayi infective-stage larva

expression library encoded a 12.5-kDa protein product (Bm-MIF) with

42% identity to human and murine MIF. MIF homologues were also found

to be expressed in the related filarial species Wuchereria bancrofti

and Onchocerca volvulus. Bm-mif was transcribed by adult and larval

parasites, and the protein product was found in somatic extracts and

in the parasite's excretory-secretory products.

Immunohistocytochemistry revealed that Bm-MIF was localized to cells

of the hypodermis/lateral chord, the uterine wall, and larvae

developing in utero. Unexpectedly, the activities of recombinant

Bm-MIF and human MIF on human monocytes/macrophages were found to be

similar. When placed with monocytes/macrophages in a cell migration

assay, Bm-MIF inhibited random migration. When placed away from cells,

Bm-MIF induced an increase in monocyte/macrophage migration that was

specifically inhibited by neutralizing anti-Bm-MIF antibodies. Bm-MIF

is the first demonstration that helminth parasites produce cytokine

homologues that have the potential to modify host immune responses to

promote parasite survival.

INTRODUCTION

Top

Abstract

Introduction

Materials & Methods

Results

Discussion

References

 

The parasitic nematodes Wuchereria bancrofti, Brugia malayi, and

Brugia timori, the etiological agents of lymphatic filariasis in

humans, infect over 120 million people worldwide. Typically,

individuals become infected in early childhood through the bite of an

infective mosquito, and in areas of endemicity the infection is

maintained for decades. The adult parasites reside in the lumen of the

lymphatics, where the females release thousands of first-stage larvae,

or microfilariae (Mf), each day into the peripheral circulation.

Although filariasis presents with a spectrum of clinical states, a

general classification defines two major groups: microfilaremic

individuals who have no discernible symptoms of infection and patients

who are amicrofilaremic and have developed chronic disease. A majority

of infected individuals are in the asymptomatic group. The immunity in

asymptomatic/microfilaremic individuals is strongly associated with a

Th2-type response with high immunoglobulin E (IgE) and IgG4 levels and

eosinophilia (30, 39, 40, 54). In contrast, the immune responses of

the amicrofilaremic/chronic pathology group are more of the Th1 type

(30, 38). Although the specific roles that Th1 and Th2 responses play

in pathology and immunity are still to be resolved, it is becoming

clear that filarial nematode development in the context of a Th2

immune response conveys an advantage for parasite survival in the

human host.

 

Among the important issues relating to parasite-host interactions is

our lack of understanding of the mechanisms that result in the

induction and maintenance of the type of immunity that accommodates

chronic, long-term filarial infections. The ability to persist in an

immunologically competent host has led to the suggestion that filarial

parasites have evolved specific measures to counter immune defenses.

In addition to anatomical and physical defenses such as size,

motility, and the presence of a thick outer covering, the cuticle,

filarial parasites produce and release as excretory-secretory (ES)

products a number of molecules that have the potential to play a role

in immune evasion. The proposed mechanisms for a number of these

putative ES-derived immune modulators, such as proteases (60),

protease inhibitors (37, 69), and antioxidant proteins (17, 36, 61),

have these molecules working locally to neutralize or to interfere

with the effector molecules of the innate and adaptive defense

responses. Whatever impact these enzymes and enzyme inhibitors have on

local effector mechanisms, it is unlikely that their actions account

for the systemic immune effects that accompany filarial infections.

One possible explanation for the strong bias toward Th2-type immunity

seen during asymptomatic filariasis is that the parasite is able to

misdirect the immune response through the presentation of epitopes

that have an inherent preference for eliciting a Th2-type response

(26) or by the elaboration of ligands and/or receptors that are

capable of altering normal signaling between cells of the immune

response. Recent reports suggest that filarial parasites of animals

have the capacity to proactively shape their immunological

environments (21, 68).

 

We report here the characterization of the first parasite-derived

homologue of a human cytokine. A gene encoding a homologue of human

cytokine macrophage migration inhibitory factor (MIF) was isolated as

a cDNA from the human filarial parasite B. malayi. MIF was originally

described as a factor that inhibited the random migration of

macrophages (12, 18). With the cloning of human and mouse MIF (46, 65)

and the development of new reagents to study this molecule, the scope

of MIF's biological activities has been significantly expanded. MIF is

constitutively expressed by T cells, macrophages, and eosinophils (5,

16, 51). It influences T-cell (5) and NK-cell (3) activation and

immunoglobulin synthesis (42) and leads to an amplification of

inflammatory responses (16). MIF also plays important roles in

endotoxin shock (4, 9), the response to glucocorticoid hormones (15),

and the regulation of insulin secretion (64), and it has been

implicated in cellular growth and differentiation events (35, 56, 66).

Interestingly, MIF has been shown to have isomerase-tautomerase

activity (7, 49, 50, 71), but the physiological substrate for this

activity has not been identified.

 

The B. malayi-derived MIF homologue (Bm-MIF) reported here was found

in both somatic extracts and the ES products of all of the stages

developing in the vertebrate host. A recombinant form of Bm-MIF was

shown to have, depending on the assay conditions, both migration

inhibitory and chemotactic activities on human peripheral

blood-derived monocytes/macrophages. Subsequent analysis demonstrated

that mif-like genes are expressed by the related filarial parasites W.

bancrofti and Onchocerca volvulus. The possible significance of

parasite-derived MIF in the immunobiology of infection, immune

evasion, and nematode biology are discussed.

 

MATERIALS AND METHODS

 

Isolation and sequencing. The clone AS3ISB220 was identified from a

B. malayi third-stage larval (L3) cDNA expression library

(JHU93SLBmL3) as part of an expressed sequence tag (EST) sequencing

initiative (11). AS3ISB220 in pBluescript (Stratagene, La Jolla,

Calif.) was sequenced completely in both directions by the fluorescent

dideoxy terminator method on an Applied Biosystems (Foster City,

Calif.) 377 automated sequencer. The DNA and deduced amino acid

sequences of clone AS3ISB220 were compared to the public protein,

nucleic acid, and EST databases by using both the BLAST (1) and FASTA

(47) algorithms. Motif analysis was carried out with the University of

Wisconsin Genetics Computer Group suite of programs (22). Clone

AS3ISB220 was designated a putative B. malayi homologue of the

mammalian cytokine macrophage migration inhibitory factor (Bm-mif).

 

RT-PCR. mRNA was isolated from 10,000 Mf, 1,000 L3s, 500 fourth-stage

larvae (L4s), or 25 adults by the Microfast Track method (Invitrogen,

San Diego, Calif.). Single-stranded cDNA was generated by reverse

transcription (RT), as recommended by the manufacturer (Stratagene).

PCR was carried out on appropriate dilutions of the templates by using

Bm-mif-specific primers (W4598 and W4599 [see below]). The Bm-mif

results were normalized to the transcriptional levels of the

constitutively expressed gene, nucleoside diphosphate kinase (Bm-ndk)

(28). Bm-ndk was amplified by using the primers XSL

(5'-GCTCTAGAGCGGTTTAATTACCCAAGTTTGAG-3') and W4353

(5'-GCTGAAGGCAAGGAATCT-3'). Following 20 cycles of amplification, the

PCR products were resolved on an agarose gel and stained with ethidium

bromide, and the gel image was digitized for densitometry analysis by

using NIH Image (developed by the U.S. National Institutes of Health

and available on the Internet at http://rsb.info.nih.gov/nih-image/).

The results for each stage were expressed as a ratio of the density of

the Bm-mif products to the density of the Bm-ndk products from the

same template.

 

Genomic DNA. Adult B. malayi nematodes were snap frozen in liquid

nitrogen, ground to a powder with a mortar and pestle, and then

suspended in 1 ml of lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM

EDTA, 1 M NaCl, 0.5% sodium dodecyl sulfate [sDS], 100 µg of

proteinase K [boehringer Mannheim, Indianapolis, Ind.] per ml, 36 mM

beta -mercaptoethanol, and 25 µg of DNase-free RNase [boehringer

Mannheim]). The genomic DNA was used as a template in PCR with primers

W4684 (5'-GAAGATCTATGCCATATTTTACG-3') and W4685

(5'-GAAGATCTTTATCCCAAAGTAGATCC-3'). The resulting PCR product was

purified (QIAquick; Qiagen, Chatsworth, Calif.), and both strands were

sequenced to completion.

 

Subcloning, expression, and purification. The sequence corresponding

to the Bm-mif open reading frame (ORF) was isolated by PCR. The 5'

primer, W4598 (5'-AGATCTGCAGCTATGCCATATTTTACGATTGATAC-3'), contained a

recognition site for PstI and 23 bp of Bm-mif ORF that included the

codon for the initiating methionine (underlined). The 3' primer, W4599

(5'-AAAAGCTTATCATCCCAAGTAGATCCATTAAAAGC-3'), contained a recognition

site for HindIII, a stop codon, and the last 23 bp of the Bm-mif ORF

(underlined). After 25 cycles of amplification, the PCR product was

subcloned in frame into pRSET B (Invitrogen), which had been digested

with PstI and HindIII. Recombinant plasmids were used to transform

Escherichia coli BL21, and the synthesis of recombinant,

histidine-tagged Bm-MIF (Bm-MIF-His) was induced with 0.4 mM IPTG

(isopropyl-beta -D-thiogalactopyranoside) for 2 h at 37°C. Purified

Bm-MIF-His was isolated from a nickel column (Ni-NTA Agarose; Qiagen)

with elution buffer (500 mM imidazole in 20 mM Tris-HCl, [pH 7.9]).

Bm-MIF-His was dialyzed against elution buffer adjusted to pH 6.0, and

the protein concentration was determined by using the bicinchoninic

acid assay (Pierce, Rockford, Ill.).

Bm-mif was also expressed as a non-fusion protein in pET11b (Novagen,

Madison, Wis.). The Bm-mif ORF was PCR amplified from pBluescript by

using the 5' primer W4690, which contained a recognition site for NdeI

and the first 15 bp of the Bm-mif ORF (underlined)

(5'-GGAATTCCATATGCCATATTTTACG-3'), and the 3' primer W4689, which

contained a recognition site for NdeI, a stop codon, and the last 15

bp of the Bm-mif ORF (underlined)

(5'-GGAATTCCATATGTTATCCCAAAGTAGA-3'). The PCR product was then cloned

in frame into the pET11b vector. Recombinant plasmids were used to

transform E. coli BL21, and recombinant Bm-MIF synthesis was induced

with 0.4 mM IPTG for 1.5 h at 37°C.

 

The purification protocol for recombinant Bm-MIF was modified from

that of Bernhagen et al. (10). Bacterial extracts were passed over a

HiTrap Q anion-exchange column (Pharmacia Biotech, Piscataway, N.J.)

followed by selective elution from a butyl-Sepharose hydrophobic

interaction column (Pharmacia Biotech). Fractions were analyzed by

SDS-polyacrylamide gel electrophoresis (PAGE) (32) and silver stained

with Silverstain Plus (Bio-Rad, Hercules, Calif.). Those fractions

deemed to be >97% pure were pooled and processed for refolding.

 

Refolding. The protocol used to generate bioactive Bm-MIF was as

described for mammalian MIF (10). Briefly, protein was denatured with

10 mM dithiothreitol (DTT) and 8 M urea (pH 6.8) for 1 h at room

temperature. Gradually, 10 mM DTT in TBS (20 mM Tris-150 mM NaCl [pH

6.8], prepared in tissue culture-grade water) was added until the urea

was diluted to 2 M. The protein was then dialyzed overnight at 4°C

against TBS with 10 mM DTT. The TBS with 10 mM DTT was gradually

replaced with TBS by dialysis at 4°C. Bioactive, lipopolysaccharide

(LPS)-free human MIF was prepared as described previously (10). Each

preparation was tested for endotoxin levels by the Limulus amebocyte

lysate chromogenic assay (BioWhittaker, Walkersville, Md.). The

preparations used had <2 pg of endotoxin/µg of protein.

 

Antisera. Anti-Bm-MIF-His and anti-Bm-MIF antibodies were produced in

mice (27). Rabbit polyclonal anti-mouse MIF and the anti-MIF

neutralizing monoclonal antibody IIID-9 have been described previously

(16).

 

Western blots. After being snap frozen in liquid nitrogen, parasites

were ground to a fine powder, resuspended in SDS-PAGE sample buffer

(0.5 M Tris [pH 6.8], 40% glycerol, 8% SDS, 4% 2-mercaptoethanol, and

0.002% bromphenol blue), incubated for 10 min at 100°C, sonicated, and

centrifuged to pellet particulate material. The parasite proteins were

separated by SDS-PAGE under reducing conditions on a 10 to 20%

acrylamide gradient, and Western blots were prepared and immunostained

as described previously (28). Extracts from O. volvulus, Ascaris suum,

Dirofilaria immitis, Schistosoma mansoni, and Caenorhabditis elegans

were prepared from frozen organisms.

 

ES products. Mf, L4 (day 15 postinfection), and adult B. malayi

organisms were obtained by lavaging the peritoneal cavity of

intraperitoneally infected male gerbils (Meriones unguiculatus) and

were washed and placed in 10 ml of Dulbecco modified Eagle medium

(GIBCO BRL Life Technologies, Grand Island, N.Y.). The Mf, L4, and

adult parasites were cultured separately at 37°C for 18 h, after which

the media were collected and processed. Media containing ES products

were centrifuged at high speed to remove particulate matter. After

addition of 1 mM EGTA, 1 mM EDTA, 2 mM PMSF (phenylmethylsulfonyl

fluoride), and 0.2 mM TLCK (Nalpha -p-tosyl-L-lysine chloromethyl

ketone) as protease inhibitors (all from Sigma), the ES products were

concentrated with an Ultrafree-MC concentrator (Millipore) with a

molecular mass cutoff of 5,000 kDa. The protein concentration was

estimated by the bicinchoninic acid protein assay (Pierce).

 

Immunohistocytochemistry. Adult B. malayi parasites were lavaged from

the peritoneal cavity of a male gerbil at 120 days postinfection,

transferred to Sorenson's buffer (4:1 0.2 M sodium phosphate

dibasic-0.2 M sodium phosphate monobasic, pH 7.4) for 1 min, and then

fixed in 4% paraformaldehyde at 4°C for 16 h. The worms were processed

for cryostat sectioning and immunostained with anti-Bm-MIF-His

antibodies by a previously described protocol (28).

 

Monocyte migration assays. The monocyte/macrophage/lymphocyte-rich

fraction of blood obtained from healthy donors was isolated by

centrifugation on a Percoll cushion (Pharmacia Biotech) (14).

Migration assays were carried out in a Micro Chemotaxis Chamber (Neuro

Probe, Cabin John, Md.) by a protocol modified from that of Schleimer

et al. (52). Briefly, wells in the bottom plate were filled with 28 µl

of PAGCM (110 mM NaCl, 5 mM KCl, 25 mM PIPES

[piperazine-N,N'-bis(2-ethanesulfonic acid)], 42 mM NaOH, 0.003% human

serum albumin, 0.1% D-glucose, 1 mM MgCl2, 1 mM CaCl2) or with

recombinant human MIF or recombinant Bm-MIF diluted in PAGCM. A

polycarbonate, polyvinylpyrrolidone-free filter containing 5-µm pores

was fitted to the bottom plate, and the top plate was secured. Cells

(50 µl of 1.8 × 106 cells/ml) suspended in PAGCM were placed in the

top wells. The chamber was incubated for 3 h at 37°C in a 5% CO2

humidified chamber. The filter was then processed for staining, and

the cells were counted under the microscope (total magnification,

×400) with the aid of an ocular grid. Each experimental condition was

replicated in three to nine wells for any one donor and repeated with

cells from three or more donors. The data are expressed as the

percentage of cells that migrated compared to that in the medium

control, which we designated 100%.

 

Nucleotide sequence accession number. The nucleotide sequence of

Bm-mif has been assigned database accession no. U88035 and assigned to

EST cluster BMC00238.

 

RESULTS

 

Clone AS3ISB220 was initially identified as part of an EST sequencing

effort (11). The 583-bp full-length insert contained the conserved

22-nucleotide spliced leader 1 (SL1) sequence trans-spliced to the 5'

end 29 bp upstream from the initiating ATG codon (see U88035). The

insert contained an ORF of 348 nucleotides and had 184 bp of 3'

untranslated sequence that included a consensus polyadenylation signal

(AATAAA) 16 bp upstream from a poly(A)17 tail.

 

When the deduced amino acid sequence was compared to the protein

sequences contained in the major databases, it was determined that

clone AS3ISB220 has significant identity to the vertebrate cytokine

macrophage MIF. At 115 amino acids, Bm-MIF is identical in size to the

described MIF proteins from human, cow, mouse, rat, and chicken (Fig.

1A). The Bm-MIF protein sequence is 40 to 42% identical and 65 to 66%

similar to the vertebrate-derived MIF sequences (Fig. 1A). In

addition, Bm-MIF contains a sequence (positions 55 through 68) that

conforms to the MIF family signature motif

[(D/E)PCA(L/V)C(V/S)LXSIGX(I/V)G].

 

 

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FIG. 1. (A) Alignment of the amino acid sequences of MIF proteins

from B. malayi (Bm-MIF [accession no. U88035]), human (hu-MIF

[accession no. 1942977]), bovine (bv-MIF [accession no. 730025]),

mouse (mu-MIF [accession no. 462602]), rat (ra-MIF [accession no.

1170956]), chicken (ch-MIF [accession no. 400257]), W. bancrofti

(Wb-MIF [accession no. AF040629]), O. volvulus (Ov-MIF [accession no.

G975442]), and C. elegans (Ce-MIF-1 [accession no. Z78012] and

Ce-MIF-2 [accession no. Z71259]). Filled areas indicate those amino

acids with identity to Bm-MIF. The amino acids that form the six beta

-strands and the two alpha -helices reported in the three-dimensional

structure of human MIF (42) are indicated by the open and hatched

boxes, respectively. The positions of the 10 invariant residues are

indicated by filled diamonds. The positions where the amino acids are

conserved in 8 of the 10 sequences are marked with open boxes. Amino

acid positions are numbered along the left margin. Percent identity

and similarity of MIF sequences to Bm-MIF are indicated at the upper

right. (B) Genomic organization of the mif genes from B. malayi

(Bm-mif), human (Hu-mif), mouse (Mu-mif), and C. elegans (Ce-mif-1 and

Ce-mif-2). Introns are shown as open boxes, and exons are filled

boxes. The size of each region (in bases) is indicated above introns

and beneath exons. Vertical bars indicate the axis of the

pseudo-twofold symmetry of the MIF protein (nucleotides 159 to 165 of

the ORF).

 

The coding region of Bm-mif was labeled and used as a probe to screen

a female cDNA library from the closely related filarial species W.

bancrofti. The full-length W. bancrofti cDNA homologue of Bm-mif,

Wb-mif, encodes 115 amino acids with 97% identity at the nucleotide

level (data not shown) and 95% identity at the amino acid level to

Bm-MIF (Fig. 1A).

 

The search of the major protein and nucleic acid databases revealed

three additional nematode-derived sequences with significant identity

to Bm-MIF (Fig. 1A). A gene identified as an expressed sequence from

L3 of a related filarial parasite of humans, O. volvulus (Ov-mif) was

found to be 26% identical and 50% similar to Bm-MIF. Two ORFs

identified in the genome of the free-living nematode C. elegans appear

to encode MIF homologues. The C. elegans gene C52E4.2, designated

Ce-mif-1, encodes a protein of 120 amino acids that is 29% identical

and 56% similar to Bm-MIF. Gene F13G3.9, designated Ce-mif-2, encodes

147 amino acids with 23% identity and 44% similarity to Bm-MIF.

 

An alignment of MIF protein sequences revealed that the amino acids at

24 positions were identical in at least 8 of the 10 MIF sequences,

with 10 of those positions being invariant (Fig. 1A). Of particular

note is the conservation of Pro at position 2, which has been shown to

be critical for isomerase function in vertebrate MIF, and the highly

conserved nature of the carboxy-terminal six residues, which are

thought to be necessary for formation of the stable MIF homotrimer (7).

 

The three-dimensional structure of the human MIF monomer has two

antiparallel alpha -helices and six beta -strands that are arranged in

pseudo-twofold symmetry (beta alpha beta beta -beta alpha beta beta )

(55). When the human MIF primary sequences corresponding to these

domains of major secondary structure were compared to the

corresponding sequences from Bm-MIF (Fig. 1A), no apparent

concentration of identical residues in these structural domains was found.

 

Genomic organization. A PCR-based strategy was used to determine the

genomic organization of Bm-mif. A 952-bp genomic fragment was obtained

and sequenced to reveal that Bm-mif contains a single 604-bp intron at

108 bases into the ORF (Fig. 1B). The intron splice site sequences

followed the GU-AG convention, with the 3' splice site conforming to

the extended 3' splice site consensus (UUUU[C/U]AG) found in C.

elegans introns (13) (see GenBank accession no. AF002699). The results

of Southern blot hybridizations indicated that Bm-mif was present in

single copy in the B. malayi genome (data not shown). A comparison of

the genomic organization of Bm-mif with vertebrate and C. elegans mif

genes demonstrated that, with the exception of the size of the first

exon of Bm-mif, human mif, and murine mif, there were no interspecies

similarities in intron or exon structure (Fig. 1B). In addition, the

genomic organization of the MIF genes did not reflect the

pseudosymmetrical domain structure of the protein. The divergent

nature of the genomic organizations suggests that MIF is an ancient

gene that has diverged through evolution or a gene that has arisen

separately in vertebrates and nematodes and gained similarity through

convergent evolution.

 

Transcription of Bm-mif. Estimates of the relative levels of

transcription of Bm-mif in the various stages of B. malayi development

were made by using semiquantitative RT-PCR. The amount of Bm-mif PCR

product for each stage was indexed to the levels obtained from a gene

known to be constitutively expressed in B. malayi, nucleoside

diphosphate kinase (Bm-ndk) (28). While all stages transcribed Bm-mif,

expression levels in the adult and Mf stages of development were

approximately twice the levels observed in L3 and L4 parasites (Fig. 2A).

 

 

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FIG. 2. (A) Transcription of Bm-mif. Poly(A)+ mRNA was isolated

from L3s, L4s, Mf, and adult (Ad) parasites, converted to

single-stranded cDNA, and used as a template in PCR with primers to

amplify Bm-mif or the constitutively expressed nucleoside diphosphate

kinase (Bm-ndk). After 20 cycles of amplification, the Bm-ndk and

Bm-mif PCR products were resolved by agarose gel electrophoresis and

stained with ethidium bromide, and densitometry was carried out on a

digitized image of the gel. To compare the levels of Bm-mif

transcription between parasite stages, the amount of Bm-mif PCR

product was indexed to the amount of Bm-ndk PCR product from the same

template. Bm-mif transcription was expressed in arbitrary units. The

data are representative of results from three independent repetitions.

(B) Bm-MIF (Bm-MIF) in extracts from L3s L4s, Mf, and Ad parasites.

Parasite proteins were separated on a 10 to 20% polyacrylamide

gradient under reducing conditions, transferred to a nitrocellulose

membrane, and immunostained. The positions of molecular mass standards

are indicated along the left margin, in kilodaltons. © Bm-MIF in ES

products. L4s, Mf, and Ad parasites were placed in culture for 18 h.

Culture medium was concentrated, separated on a 15% polyacrylamide gel

under reducing conditions, transferred to a nitrocellulose membrane,

and immunostained with anti-Bm-MIF-His antibodies. Recombinant Bm-MIF

(Bm MIF) and an adult somatic antigen (Ad som) extract were included

as positive controls. (D) Detection of MIF-like proteins in extracts

from B. malayi (Bm), O. volvulus (Ov), A. suum (As), D. immitis (Di),

S. mansoni (Sm), and C. elegans (Ce). Protein extracts were separated

on a 15% polyacrylamide gel under reducing conditions, transferred to

a nitrocellulose membrane, and immunostained with anti-Bm-MIF-His

antibodies.

 

Bm-MIF. Bm-mif was expressed as a histidine-tagged fusion protein

(Fig. 3). Mouse anti-Bm-MIF-His antibodies were used to immunostain

Western blots containing equal amounts of protein from staged B.

malayi parasites to evaluate the nature of parasite-derived Bm-MIF

(Fig. 2B). Two bands were resolved in extracts of L4s, Mf, and adults,

with estimated molecular masses of 12.3 and 12.8 kDa. Although only

the 12.3-kDa band was resolved in extracts of L3 parasites here, both

bands were resolved when more L3 protein was placed on the blot (data

not shown). The presence of two potential N-linked glycosylation sites

suggested that one explanation for the two bands could be differential

glycosylation. However, treatment of B. malayi adult extracts with

endoglycosidase F resulted in no shift in the pattern of antibody

recognition, indicating that N-linked glycosylation does not account

for the two immunoreactive bands (data not shown).

 

 

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FIG. 3. Purification of recombinant Bm-MIF. Lanes 1 to 3, pRSETB.

Protein extracts from induced bacteria carrying pRSETB with no insert

(lane 1) and from induced bacteria carrying pRSETB-Bm-mif (lane 2) are

shown. The induced extract was passed over a nickel affinity column,

and the 16.5-kDa His-tagged Bm-MIF (Bm-MIF-His) fusion protein was

eluted with imidazole (lane 3). Extracts were separated on a 15%

polyacrylamide gel under reducing conditions and visualized with

Coommassie blue staining (top). The proteins from an identical gel

were transferred to a nitrocellulose membrane and immunostained with

anti-Bm-MIF-His (bottom). Lanes 4 to 7, pET11b. Protein extracts from

induced bacteria carrying pET11b with no insert (lane 4) and from

induced bacteria carrying the pET11b-Bm-mif construct (lane 5) are

shown. The extracts of induced bacteria were passed over a MonoQ

column (lane 6), and the flowthrough was placed on a butyl-Sepharose

column. The 12.5-kDa Bm-MIF protein was eluted from the

butyl-Sepharose with decreasing amounts of salt (lane 7). Proteins

were separated on a 15% polyacrylamide gel under reducing conditions

and visualized with silver staining (top). The proteins from an

identical gel were transferred to a nitrocellulose membrane and

immunostained with anti-Bm-MIF-His (bottom).

 

Adults, Mf, and L4s were placed in culture and the media were

collected, concentrated, and analyzed by immunoblotting to determine

if Bm-MIF was a component of the parasite's ES products. Bm-MIF was

detectable in the ES products from all of the stages tested (Fig. 2C).

It was estimated that Bm-MIF makes up approximately 1.5% of the ES

proteins released by adult parasites (data not shown).

 

Immunoblot analysis was used to determine if the anti-Bm-MIF-His

antibodies could detect Bm-MIF-like molecules in extracts from other

nematode and helminth parasite species. Proteins with an estimated

molecular mass of 12.3 kDa were present in extracts of the parasitic

species O. volvulus, A. suum, and D. immitis but not in extracts of

the digenetic trematode S. mansoni (Fig. 2D). Under the conditions

used here, anti-Bm-MIF-His antibodies did not detect putative MIF

homologues from C. elegans.

 

Immunohistocytochemistry. Anti-Bm-MIF-His bound strongly to the

uterine lining and to the noncellular material associated with the

developing embryos in sections of gravid female parasites (Fig. 4a).

In addition, Bm-MIF was localized to the hypodermis and to the surface

of the major body wall muscle bundles. In sections of females where

more-developed embryos were evident, the embryos showed low levels of

staining (Fig. 4d). In sections of male parasites, antibody staining

was restricted to cells of the hypodermis/lateral chord (Fig. 4b).

 

 

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FIG. 4. Immunohistocytochemical localization of Bm-MIF in female

(a, c, and d) and male (b) B. malayi organisms. Cryostat sections were

immunostained with mouse anti-Bm-MIF-His antibodies (a, b, and d) or

normal mouse serum © followed by a biotinylated horse anti-mouse

antibody. Antibody binding was resolved with the Vector ABC

immunostaining kit. g, intestine; h, hypodermis; m, somatic muscles;

t, testis; u, uterus. Under the conditions used here, the intestine

stained nonspecifically.

 

Migration assays. Initial studies demonstrated that amino acid tags on

either the N or the C termini of recombinant Bm-MIF resulted in the

production of molecules with no bioactivity. In order to produce a

recombinant protein with bioactivity, we prepared constructs to

produce Bm-MIF as a non-fusion polypeptide in pET11B (Fig. 3). Prior

to use in the migration assays, the purified recombinant Bm-MIF was

denatured with DTT and urea and then gradually refolded to an active

conformation by dialysis.

One standard for bioactivity in murine and human MIF is its ability to

inhibit random migration of macrophages in an in vitro assay.

Therefore, we established a migration assay to determine if Bm-MIF had

any direct action on human peripheral blood monocytes/macrophages and

to test the hypothesis that Bm-MIF would alter the action of human

MIF. The results of migration assays produced two unexpected results.

First, in our initial studies with human MIF, we found that while

recombinant human MIF did inhibit random migration when placed in the

top chamber with the peripheral blood monocytes/macrophages, when it

was placed in the bottom chamber of the apparatus it functioned as a

chemoattractant (Fig. 5A). The ability of human MIF to induce

chemotaxis was specifically inhibited in a concentration-dependent

fashion by an anti-human MIF monoclonal antibody (Fig. 5C). Therefore,

depending on the specific circumstances, human MIF can inhibit or

enhance chemotaxis of human monocytes and macrophages.

 

 

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FIG. 5. Bm-MIF (Bm-MIF) and human MIF (hu-MIF) induce changes in

macrophage migration. Human peripheral blood monocytes/macrophages

were placed in the top well of a microchemotaxis chamber, as outlined

in Materials and Methods. The level of migration of cells exposed to

human MIF (A and C), Bm-MIF (B and D), or medium was assessed after 3

h of incubation at 37°C. The level of migration of cells in medium

alone was designated 100% and is indicated by the arrow in each panel.

Each series of test and control treatments was carried out in

triplicate on cells isolated from three to six healthy donors. Error

bars represent standard errors of the means for the individual donors.

(A) Human MIF at three concentrations in the top wells or the bottom

wells of the chemotaxis chamber. (B) Bm-MIF at various concentrations

in the top wells or the bottom wells of the chemotaxis chamber. ©

Specific inhibition of human MIF-induced migration with monoclonal

antibody IIID-9. The migration induced by 1 ng of human MIF per ml in

the bottom chamber is shown by the first bar and by the dashed line.

IIID-9 or its corresponding isotype-matched control monoclonal

antibody (Ctrl IgG) was preincubated with 1 ng of human MIF per ml for

1 h at 25°C and then placed in the bottom chamber. Reagent control

reaction mixtures included cells incubated with IIID-9 or the isotype

control IgG only under identical culture conditions (eighth and ninth

bars, respectively). (D) Specific inhibition of Bm-MIF-induced

migration of human monocytes/macrophages by anti-Bm-MIF polyclonal

sera. The migration induced by 1 ng of Bm-MIF per ml in the bottom

chamber of the apparatus is shown by the first bar and by the dashed

line. Anti-Bm-MIF serum or preimmunization control serum (Ctrl Ab) was

preincubated with 1 ng of Bm-MIF per ml for 1 h at 25°C prior to

placement of the mixture in the bottom chamber. Reagent control

reaction mixtures included cells incubated with anti-Bm-MIF or

preimmune serum only under identical culture conditions (eighth and

ninth bars, respectively).

 

The second unexpected result was found in migration assays using the

parasite-derived MIF. Bm-MIF had an effect on human

monocytes/macrophages that was nearly identical to that of human MIF.

When placed in the top chamber with the cells, it inhibited migration

by ~50 to 75%, and when placed in the bottom chamber, Bm-MIF enhanced

migration (Fig. 5B). This activity of Bm-MIF was also inhibited in a

concentration-dependent fashion by a mouse anti-Bm-MIF antibody (Fig.

5D). It is important to note that LPS in the recombinant MIF

preparation was below 2 pg/µg of protein and that, in this assay

system, LPS in the bottom wells actually inhibited migration (data not

shown).

 

To test the possibility that interactions of Bm-MIF with host-derived

MIF on the same cell lead to altered activity, Bm-MIF plus human MIF

were placed in the bottom wells of the migration chambers. Together,

the two cytokines induced the same level of monocyte/macrophage

migration as seen when the cells were exposed to only one of the

molecules (data not shown).

 

DISCUSSION

Top

Abstract

Introduction

Materials & Methods

Results

Discussion

References

 

In order to survive immune attack, pathogens have adopted a variety of

strategies to evade or modify immune responses. There is an increasing

appreciation that one of the approaches used by pathogens is to

produce homologues of host molecules that are important in immune

signaling to blunt or divert inflammation. This approach has been best

documented with viruses. Poxviruses secrete chemokine-like molecules

and chemokine binding factors that result in altered trafficking of

infiltrating leukocytes into areas of virus infection (31, 33).

Cytomegalovirus blocks the ability of the acquired and innate immune

systems to recognize infected cells by interfering with the expression

of host class I major histocompatibility complex molecules and

deploying a virus-encoded class I-like molecule (24). A number of

viruses, including Epstein-Barr virus, produce an interleukin 10

homologue that presumably functions in altering antiviral responses

(25, 70). We present here the first example of a parasite-derived

molecule with significant homology to a human cytokine that functions

to alter the behavior of human cells.

 

The results of in vitro macrophage migration assays indicate that

Bm-MIF is chemotactic for human cells. Assuming that Bm-MIF retains

this function in vivo, it raises the question of why a parasite would

release a molecule that functions in attracting cells important in

immune signaling and defense. It is possible that the parasites

attract host macrophages as the first step in a process that leads to

alterations in their induction and/or effector functions. Bm-MIF or

other ES molecules may change the levels of certain cytokines produced

by antigen-presenting cells, creating an immunological environment

that promotes parasite survival. This may, in part, explain the strong

Th2 bias seen in a majority of chronically infected individuals (30,

38, 40, 54). Another potential reason for manipulation of the cytokine

profile may be to obtain host-derived factors necessary for parasite

development. Cytokines have been shown to be essential growth and

reproductive cues in both protozoan (6) and helminthic parasites (2).

 

Although no additional parasite-derived cytokine homologues have been

identified, parasites have been shown to produce factors that modify

immune responses. Both Leishmania spp. and Trypanosoma cruzi release

proteins that change the levels of cytokine expression of human

macrophages and dendritic cells (20, 48). African trypanosomes secrete

a molecule that selectively induces CD8+ T cells to secrete gamma

interferon (62). Recent reports suggest that ES products from filarial

parasites of animals have the capacity to proactively shape their

immunological environment as well. A 62-kDa glycoprotein released by

the rodent filarial parasite Acanthocheilonema viteae interferes with

antigen receptor-mediated activation of B cells and T cells (21). A

factor isolated from the ES products produced by the major filarial

parasite of dogs, D. immitis, increases receptor expression on T and B

cells, Th2 cytokine production, and IgE synthesis (68). In addition,

evidence is accumulating that suggests that both protozoan (44) and

helminthic (19, 29) parasites exploit the transforming growth factor

beta serine-threonine kinase receptor-ligand system to promote

parasite survival and development.

 

Over 30 years ago, one of the first lymphokine activities described

was a soluble factor elaborated by activated T cells that had the

ability to inhibit random migration of macrophages (12, 18). With the

cloning of genes encoding human and mouse MIF (46, 65), its perception

as a T-cell-derived molecule that simply functions to inhibit

macrophage migration has been expanded to an appreciation that the

sources of MIF are diverse and its actions are complex. In addition to

T cells, macrophages have been shown to be both an important target

for and a major source of MIF (16). MIF is also produced by

eosinophils (51), the corticotrophic cells of the anterior pituitary

gland (9, 10), the beta cells of the islets of Langerhans (64), the

differentiating cells of the eye lens (66), and the cells of the basal

layer of human epidermis and keratinocytes (53). The association of

MIF with a variety of cell types in vertebrates suggests that it may

carry out multiple functions.

 

As the cellular sources of MIF have become increasingly complex, so

too have the MIF-related immune functions. MIF has direct effects on

T-cell activation and antibody production (5). The T-cell-derived

glycosylation inhibition factor that regulates IgE synthesis by B

cells has been shown to be identical to MIF (42). MIF has been

associated with the macrophage infiltrates in delayed-type

hypersensitivity lesions (8), glomerulonephritis (34), rheumatoid and

collagen-induced arthritis (41, 43), and acute respiratory distress

syndrome (23). Preformed MIF is released from macrophages (16) and

cells of the anterior pituitary (9) into the circulation in response

to LPS and mediates an upregulation of tumor necrosis factor alpha

production (9, 16). Glucocorticoids also induce the release of MIF

from macrophages and from T cells, where it functions to override the

strong suppressive action that steroids have on T-cell proliferation

and cytokine production (4). MIF has been shown to inhibit

NK-cell-mediated cytotoxicity by preventing the release of perforin

(3). These observations strongly implicate MIF as an important factor

in disease pathogenesis.

 

Of particular importance here is the demonstration of a role for MIF

activity in the context of parasitic diseases. Macrophages secrete

large amounts of MIF after phagocytosing malaria-infected erythrocytes

or malaria hemazoin, and there are increased circulating levels of MIF

during Plasmodium chabaudi infections in mice (41). Treatment of

murine macrophages in vitro with MIF significantly enhances their

ability to kill Leishmania major (45), and administration to mice in

vivo significantly reduces disease (67). It is likely that host MIF

also plays an important role in regulating filarial infections.

 

The association of vertebrate MIF with cells undergoing growth and

differentiation suggests that it may play a role in normal cell

biology. MIF has been identified as an important protein in rapidly

differentiating cells (35, 53, 66) and in embryos (57, 63). The

localization of Bm-MIF to multiple cell types in B. malayi (Fig. 3)

and the presence of two MIF-like genes from the free-living nematode

C. elegans suggest that the nematode-derived molecules may have

important actions on nematode cells. Further work has demonstrated

that both Ce-mif-1 and Ce-mif-2 are transcribed (data not shown). The

presence of MIF homologues in C. elegans provides a well-defined and

highly manipulatable system to characterize the roles that MIF

proteins may have in the cell biology of nematode development.

 

MIF is distinct from other cytokines in that it also catalyzes

chemical reactions. It has been shown to have tautomerase activity on

at least two substrates (49, 50). Although the native cellular

substrates(s) is not known, it is possible that at least part of the

bioactivity of MIF is mediated through its ability to tautomerize. We

have shown that Bm-MIF also has tautomerase activity (data not shown),

although the level of activity is significantly lower than mammalian

MIF when assayed with the currently known substrates. The tautomerase

activity of MIF is dependent on the proper folding of the molecule and

the presence of the conserved residues at position 2 (Pro) and at the

C terminus of the molecule (7), all of which are present in Bm-MIF.

Crystallographic studies of recombinant human MIF (55, 58) have shown

that the monomer contains two antiparallel alpha -helices that pack

against a four-stranded beta -sheet and assemble into a barrel-shaped

homotrimer with a solvent-accessible channel that runs the entire

length of the threefold axis (55). The C-terminal residues are

important in intersubunit interactions that lead to a stable

homotrimer (7, 55). The N-terminal proline is positioned on the

outside of the barrel and is believed to be the active residue during

catalysis. Interestingly, mapping of the 10 invariant residues found

in all members of the MIF family onto the three-dimensional structure

of human MIF shows that most of these residues cluster around the

N-terminal proline, forming a pocket around the N-terminal proline

that resembles an enzymatic active site (59).

 

A parasite-derived MIF homologue that is capable of diverting or

modifying important functions of the host-derived molecule could

contribute significantly to the parasite's ability to survive and

replicate. Locally, this could result in diminished or qualitatively

altered inflammatory responses that provide the parasite with a

short-term survival advantage. Systemically, changes in normal MIF

signaling may be one of the determining factors for the strong type 2

bias of the immune response observed in chronic filarial infections

(30, 39, 40). A better understanding of the nature of these molecules

and how they function at the level of the host cell to alter immune

responsiveness will be critical for the development of effective

therapies and for understanding pathogenesis in humans.

ACKNOWLEDGMENTS

 

This work was supported by research grants from the U.S. Public Health

Service (AI-29411), the Edna McConnell Clark Foundation (EMCF 01093),

and the World Health Organization (T23/181.80). Parasites and infected

gerbils were supplied under the auspices of an NIAID supply contract

(AI-02642), U.S./Japan Cooperative Medical Science Program.

 

We thank Brian Schofield for valuable assistance in carrying out

immunohistocytochemistry and M. Blaxter for sequencing and bioinformatics.

FOOTNOTES

 

* Corresponding author. Mailing address: Department of Molecular

Microbiology and Immunology, School of Hygiene and Public Health,

Johns Hopkins University, 615 North Wolfe St., Baltimore, MD 21205.

Phone: (410) 955-3442. Fax: (410) 955-0105. E-mail: ascott.

 

Editor: J. M. Mansfield

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