A new mechanism for IL-5-dependent helminth control: neutrophil accumulation and neutrophil-mediated worm encapsulation in murine filariasis are abolished in the absence of IL-5

Khaled M. Al-Qaoud, Eric Pearlman1, Thomas Hartung2, Jan Klukowski, Bernhard Fleischer and Achim Hoerauf

Bernhard-Nocht-Institute of Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany
1 Division of Geographic Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
2 Department of Biochemical Pharmacology, University of Konstanz, 78434 Konstanz, Germany

Correspondence to: A. Hoerauf


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-5 production and eosinophilia are features of helminth infections, but results concerning the role of IL-5 and eosinophils (EP) in worm control are contradictory. We describe here a novel, IL-5-dependent mechanism of helminth control in vivo, using a fully permissive murine filariasis model, i.e. infection of BALB/c mice with Litomosoides sigmodontis. Worm control was exerted by the formation of inflammatory nodules around adult filariae which initially remained alive but were eventually killed within several weeks. The cell population essential for inflammatory nodule formation was found to be neutrophils (NP) but not EP. Neutralization of IL-5 led to a failure of both EP and NP accumulation at the site of infection (i.e. the thoracic cavity), resulting in cessation of inflammatory nodule formation around worms and in their survival. The role of NP in this process was confirmed by treatment of mice with anti-granulocyte colony stimulating factor (G-CSF) which also resulted in a lack of inflammatory nodule formation and worm killing albeit in the presence of EP. Since IL-5, due to the absence of IL-5 receptors on NP, does not act on these cells directly, it was investigated if anti-IL-5 altered the production of NP-chemotactic cytokines. In anti-IL-5-treated mice, cytokines known to promote NP accumulation like tumor necrosis factor-{alpha}, G-CSF and KC (IL-8) were found to be strongly reduced, while NP-deactivating cytokines like IL-10 were increased. In conclusion, IL-5 constitutes a cytokine essential for NP-mediated worm control in filarial infection.

Keywords: animal model, cytokines, eosinophils, helminth infection, neutrophils


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Infections with helminths are characterized by high IgE responses and increased numbers of circulating eosinophils (EP). EP are known to be capable of killing helminths upon activation in vitro (1,2). However, experiments in vivo aimed at investigating the role of EP or the EP-activating cytokine IL-5 against helminth infections were not unequivocal. Thus, treatment of helminth-infected mice with monoclonal anti-IL-5 antibody abolished protective immunity or non-permissivity in some infections (35) but did not change the course of disease in others, e.g. Schistosomiasis (6) and gastrointestinal nematode infections (for review, see 7).

To date, due to the lack of a suitable model, no studies have detailed the involvement of IL-5 and EP by an interventional approach in a fully permissive filarial infection in laboratory mice. A complete filarial infection cycle was recently established in BALB/c mice using the rodent filaria Litomosoides sigmodontis (8). In this infection, infective L3 are transmitted from mites and migrate to the thoracic cavity; here they mature and after 6 weeks produce microfilariae (mf) which can be found in the blood stream. Our earlier studies using this model showed high levels of IL-5 and eosinophilia in the thoracic cavity, present already shortly after L. sigmodontis L3 migration and persisting through all developmental stages (9,10). Hypothesizing that EP could play a role in the destruction and elimination of this parasite, we analyzed the course of filarial infection in BALB/c mice treated with anti-IL-5. Our results show that neutrophils (NP) but not EP are important effector cells against the adult filariae, being prevented from accumulation in the absence of IL-5.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animal maintenance, antibody treatment and L. sigmodontis infection
BALB/c mice (originally from Charles River, Sulefeld, Germany) and cotton rats were bred at the animal facilities of the Bernhard-Nocht-Institute. Natural infections of mice with L. sigmodontis were performed as described previously (9,10). At least seven mice were used for each treatment group.

mAb were purified from hybridoma culture supernatants [TRFK-5, anti-mouse IL-5; TRFK-4, control isotype; NIMP-R14, anti-mouse NP (11)] using ammonium sulfate precipitation and affinity purification over a Protein G column (Pharmacia, Freiburg, Germany) according to standard procedures. The mAb batches were free from other proteins as judged by PAGE analysis. Anti-granulocyte colony stimulating factor (G-CSF) polyclonal IgG antibodies (12) were also purified from goat sera using Protein G–Sepharose. Mice were treated with 500 µg/ml anti-IL-5 or control mAb, 4 days before L. sigmodontis infection and every 3 weeks thereafter. This dose was found previously to deplete EP for 4 weeks after each injection. Treatment with anti-G-CSF antibody (3 mg/mouse/dose) was started at day 43 post-infection, i.e. 5 days before the onset of microfilaremia, and continued at weekly intervals thereafter.

Parasite and inflammatory nodule recovery
The number of adult worms was counted at days 28 and 80 post-infection To this, the thoracic cavity, the representative site for assessment of worm numbers (13), was flushed with 10 ml PBS/1% FCS and worms were allowed to sediment. The sediment was also used to determine the number of inflammatory nodules. Microfilaremia was determined as described (9).

Cytospin preparations
An aliquot of 200 µl of thoracic cavity cells (2x105/ml in PBS/1% FCS) was centrifuged against a glass slide with absorptive filter paper using a Shandon cytocentrifuge. Cytospins were either used for Giemsa staining or were fixed in acetone, air-dried and stored at –70°C for staining of intracellular cytokines.

EP count in peripheral blood and thoracic cavity exudate
EP in pleural exudate cells (PLEC) were enumerated by staining with Hinkelmann's solution and counted using a Neubauer hemocytometer (9). Cytospin slides were stained with Wright–Giemsa stain (Sigma, Taufkirchen, Germany) and differentially enumerated.

Histology and immunohistochemistry
Inflammatory nodules were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 µm) were cut using a microtome (Reichert-Jung, Hamburg, Germany). Sections were then cleared in xylin for 5 min, rehydrated in a decessive dilution of ethanol and washed with aqua bidest.

To detect EP and major basic protein (MBP), paraffin sections were incubated with rabbit antisera to murine MBP at 1:1000 dilution in 1% FCS in 0.05 M TBS at room temperature in a humidified chamber for 2 h. Thereafter, biotinylated goat anti-rabbit Ig (Dako, Carpenteria, CA), diluted 1:200 in 1% FCS in 0.05 M TBS, was added for 30 min (14). To detect NP, paraffin sections were incubated with anti-NP mAb NIMP-R14 [rat IgG 2b (15); 100 µl of a 100 hybridoma culture supernatant containing 100 µg/ml protein], followed by biotinylated goat anti-rat Ig (5 µg/ml).

Slides were further incubated with prediluted AP-conjugated streptavidin (BioGenex, San Ramon, CA). Positive reactivity was visualized using Vector Red Substrate containing 12 mg Levamisole (Sigma) and counterstaining with modified Harris' hematoxylin (Richard-Allen, Kalamazoo, MI).

Acetone-fixed PLEC cytocentrifuge preparations were permeabilized with 0.1 % saponine and stained with biotinylated anti-IL-4, anti-tumor necrosis factor (TNF)-{alpha}, anti-granulocyte macrophage colony stimulating factor (GM-CSF) (4 µg/ml) mAb or with isotype control mAb (IgG1 or IgG2b; PharMingen) for 90 min. After a 10 min wash in two changes of PBS/0.1% saponin, the slides were incubated with phycoerythrin-conjugated streptavidin (1:50; PharMingen). This was followed by incubation in 100 µl 0.1% FITC (Sigma) in PBS, making use of the fact that FITC binds with the basic proteins in the eosinophilic granules (16,17) so that EP can be visualized.

Cell culture
The culture of thoracic cavity macrophages (MP) was carried out after adhesion on tissue culture Petri dishes (Greiner, Frickenkausen, Germany) or 96-well culture plates (Greiner) in RPMI/5% FCS at 37°C and 5% CO2 for 2 h. Since there were differences in the cellular composition between control and anti-IL-5-treated mice (more MP in the latter, see Fig. 1Go), we first did a FACS analysis to determine the relative proportion of MP in the thoracic cavity cells. According to these data, the cell input was calculated such that 30,000 MP/well were allowed to adhere. The non-adherent cells were removed by washing 3 times with PBS. Adherent cells were cultured for 24 h in the presence of medium or lipopolysaccharide (100 ng/ml). Purity was always >97% (not shown).




View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Neutralization of IL-5 prevents both EP and NP migration. Accumulation of EP (A) and NP (B) granulocytes in the thoracic cavity of L. sigmodontis-infected BALB/c mice, treated with control-Ig or with anti-IL-5 antibodies. Non-infected BALB/c mice are shown for comparison. Data are based on cytospin evaluation of thoracic cavity cells from seven mice per treatment group, using Wright–Giemsa stain and on FACS analysis. The two major other groups of cells in the thoracic cavity, as analyzed by FACS, were MP (Mac-1+, C) and B cells (B220+, D). The remainder comprised CD4+ T cells (3–7%), CD8+ T cells (1–2%) and cells not stained with either of these markers (5–10%). Data are from one representative out of three consistent experiments. In this experiment, cell counts in the thoracic cavity were as follows: non-infected mice, 0.7 ± 0.3x106; infected, control Ig-treated mice, 13.7 ± 6.4x106 (day 28 post-infection), 31.3 ± 7.2x106 (day 80 post-infection); infected, anti-IL-5-treated mice, 7.8 ± 2.9x106 (day 28 post-infection), 14.7 ± 3.3x106 (day 80 post-infection).

 
Cytokine assays
Cytokine concentrations (IFN-{gamma}, IL-4, IL-10, IL-12, TNF-{alpha} and GM-CSF) in supernatants of MP cultures and/or pleura exudates (thoracic wash) were determined by specific two-site ELISAs using standard protocols. The antibody pairs for capture and detection (biotinylated) were purchased from PharMingen in the combination recommended. Recombinant cytokines (PharMingen and R&D Systems, Wiesbaden, Germany) were used as standards. Mouse KC (homologue of human IL-8) was determined using affinity-purified goat IgG as capture and as detector (R & D, cat. no. AF-453-NA and BAF-453). All ELISAs using biotinylated secondary antibodies were developed after incubation with streptavidin–peroxidase complex (1:10,000; Boehringer, Mannheim, Germany), using 3,5,3',5'-tetramethylbenzidine as substrate (Roth, Karlsruhe, Germany; dissolved 6 mg/ml in DMSO). Sensitivity was 1 pg/ml for all cytokines except for IL-12 (12.5 pg/ml).

Reverse transcription and competitive PCR assay
Total mRNA from homogenized whole lungs was isolated by phenol–chloroform extraction and transcribed to cDNA using standard protocols established in our laboratory (18). Semiquantitative PCR for TNF-{alpha} was performed as described (19), using a plasmid which contains sequences competing for the primers used to amplify ß-actin as well as various cytokines (18). In brief, in a first PCR, aliquots of cDNA were assayed for levels of ß-actin (5' sense primer: ATG GAT GAC GAT ATC GCT; 5' anti-sense: ATG AGG TAG TCT GTC AGG T) by placing a fixed concentration of the target cDNA samples against serial (1:2) dilutions of competitor plasmid. In a second PCR the same procedure was carried out using primers specific for TNF-{alpha} (5' sense: GTC TAC TTT GGA GTC ATT GC; 5' anti-sense: GAC ATT CGA GGC TCC AGT G). Reactions were carried out for 35 cycles for both products in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). An index was calculated between the dilution at equivalence for TNF-{alpha} over that of ß-actin.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-IL-5 treatment does not affect early adult worm development but leads to higher worm survival during patency
Neutralization of IL-5 by anti-IL-5 mAb depleted EP from the peripheral blood of mice during the infection period analyzed (data not shown). In addition, the marked and early infiltration of EP in the thoracic cavity observed in infected control mice was prevented by anti-IL-5 treatment (Fig. 1AGo). However, there was no significant effect on the worm load at day 28 post-infection (Table 1Go). This indicates that the development from L3 to adult worms, i.e. worm establishment, was not influenced by this treatment. In contrast, a major effect of anti-IL-5 treatment was observed at a late stage of infection (day 80 post-infection). Whereas in normal mice, adult live worms had decreased in number by that time, there was no such decline in anti-IL-5-treated animals (Table 1Go). Consistent with the higher worm load, anti-IL-5-treated mice displayed up to 10-fold higher microfilaremia (266 ± 207 mf/50 µl blood in anti-IL-5-treated versus 22 ± 4.5 mf/50 µl blood in control mice, P < 0.05) and 5-fold higher levels of thoracic cavity mf (1420 ± 531 versus 281 ± 236 mf, P < 0.04, in anti-IL-5-treated and control mice respectively).


View this table:
[in this window]
[in a new window]
 
Table 1. Worm load and frequency of inflammatory nodule formation in L. sigmodontis-infected BALB/c mice treated with anti-IL-5 or anti-G-CSF
 
Anti-IL-5 treatment blocks formation of inflammatory nodules around worms
The decline of live worms was found to be secondary to an encapsulation process by inflammatory tissue (inflammatory nodule, Fig. 2AGo). This process began around week 7 post-infection, continued for a further 4–5 weeks while the worms were still alive and motile with one free end (see arrowhead in Fig. 2AGo), but ultimately led to worm absorption and elimination by week 20 post-infection (data not shown). The formation of these inflammatory nodules was found to be 8.5-fold reduced in mice treated with anti-IL-5 (Table 1Go). The number of inflammatory nodules relative to the live worms (index) showed a 17-fold lower frequency of inflammatory nodule formation in anti-IL-5-treated compared to normal mice (Table 1Go). In addition, the volume of the inflammatory nodules in anti-IL-5-treated mice was up to 25 times smaller (18 ± 21 mm3 in anti-IL-5-treated versus 440 ± 400 mm3 in control mice).



View larger version (96K):
[in this window]
[in a new window]
 
Fig. 2. Formation of inflammatory nodules by NP and production of NP-chemotactic TNF-{alpha} are inhibited in the absence of IL-5. Data are from one of three consistent experiments, using seven mice per treatment group and more than five different nodules per mouse. (A) Live female adult worm (left) and worm encapsulated by inflammatory tissue (right), day 80 post-infection. Note the free end of the worm (black arrowhead) which was still motile. (B) Thoracic cavity cell cytocentrifuge preparation (Wright–Giemsa stain, x600) from 80 day infected, control Ig-treated mouse. Besides mononuclear cells, both EP (black arrowhead) and NP (blue arrowhead) granulocytes are seen. (C) Microscopical analysis of transverse section of adult worm encapsulated in an inflammatory nodule, immunostain using a rabbit anti-MBP antiserum, anti-rabbit biotin antibodies and AP-conjugated streptavidin (x220). MBP+ (red) EP (yellow arrowhead) are observed scattered in the inflammatory tissue but not located adjacent to the worm. (D) Transverse section of adult worm in an inflammatory nodule, immunostain using NIMP-R14 (anti-NP), anti-rat biotin antibodies and AP-conjugated streptavidin (x220). The innermost layer of cells around the worm is formed by NIMP-R14+ (red) NP granulocytes (yellow arrowheads). (E and F) Same as (C) and (D) respectively, larger magnification (x400). (G) Thoracic cavity cell cytocentrifuge preparation from 80 day infected, control Ig-treated mouse, staining with FITC and anti-TNF-{alpha} plus phycoerythrin–anti-rat antibodies (x600). Bright-green EP are seen forming clusters with MP which stain positive for TNF-{alpha}. (H) Same as in (G), cell preparation from an anti-IL-5-treated mouse. There are no EP, MP are negative for TNF-{alpha}.

 
NP but not EP infiltrate around worms in the patent stages in infected control mice
Histological analysis of inflammatory nodules was carried out to evaluate a possible participation of EP in inflammatory nodule formation. Polymorphonuclear cells infiltrated around the encapsulated worms in inflammatory nodules from day 80 post-infection. However, staining for the EP marker MBP showed that EP were not located adjacent to the worm but behind an inner layer of other granulocytes (Fig. 2CGo, yellow arrowhead, and E). Staining with a monoclonal anti-NP antibody (NIMP-R14) revealed that these granulocytes around the worms were NP (Fig. 2DGo, yellow arrowheads, and F). In nodules from anti-IL-5-treated animals, immunohistology using either anti-NP or anti-MBP antibody did not reveal major differences in the nodule architecture compared to control animals (not shown).

NP accumulation is blocked by anti-IL-5 as well as by anti-G-CSF
Given that NP formed the inner layer of inflammatory nodules, we next investigated whether these cells were also detectable free in pleural exudate, using cytospins stained with Wright–Giemsa (Fig. 2BGo). These analyses revealed that NP were not present in the thoracic cavity before the onset of microfilaremia (day 45–50 post-infection). However, thereafter they increased in numbers throughout the observation period (Fig. 1BGo). In contrast, anti-IL-5-treated mice had 10-fold fewer NP in the thoracic cavity (Fig. 1BGo). The numbers of NP in the peripheral blood of anti-IL-5-treated mice was normal (not shown), indicating that the absence of IL-5 affected the migration to the infection site rather than the maturation of NP in the bone marrow. NP were not observed in the thoracic cavity of non-infected mice.

These data show that anti-IL-5 inhibits NP accumulation at the site of infection and, thus, the formation of inflammatory nodules. Importantly, depletion of NP with antibodies against G-CSF confirmed their role in nodule formation: although these antibodies could only be applied twice (due to host reaction) and did not completely eliminate NP from the thoracic cavity (5.5% of total PLEC in treated versus 15% in normal infected mice), this treatment reduced the numbers of inflammatory nodules 4-fold (Table 1Go), the nodules being 10 times smaller in volume. Anti-G-CSF-treated mice also displayed a 3-fold higher worm number and a 20-fold lower nodule formation index after 80 days of infection (Table 1Go). EP and MP were not reduced in numbers in the thoracic cavities of anti-G-CSF-treated mice (not shown).

NP chemotactic cytokines are diminished after anti-IL-5 treatment
NP have been described not to express IL-5 receptors (summarized in 20). Therefore, in search of an indirect pathway of NP activation controlled by IL-5, we analyzed cytokines with NP-chemotactic activity from the thoracic cavities and sera of non-infected mice and of mice at day 80 post-infection.

In infected control mice TNF-{alpha} levels were highly elevated in the thoracic cavity; serum levels were 3- to 4-fold lower, suggesting that TNF-{alpha} found in the serum originates from the thoracic cavity (Table 2Go). In contrast, both thoracic cavity and serum TNF-{alpha} levels in anti-IL-5-treated mice remained at background levels. G-CSF is another cytokine known to promote NP generation and function, and we had shown it to be essential for inflammatory nodule formation (Table 1Go); this cytokine was found in 5-fold higher levels in the thoracic cavity of control-infected compared to anti-IL-5-treated mice (Table 2Go). Several other cytokines with known NP-chemotactic activity such as KC (murine homologue of IL-8), GM-CSF and IL-4 were also elevated in infected control mice (albeit less than TNF-{alpha}) but reduced or abolished in anti-IL-5-treated mice (Table 2Go). In addition, IFN-{gamma} but not IL-12 levels were found to be diminished after IL-5 treatment. In contrast to the NP-chemotactic cytokines above, the main source of IFN-{gamma} and IL-12 appears to be not within the thoracic cavity, since levels 30-fold higher than the respective levels in the thoracic cavity were found in sera of mice (Table 2Go). A kinetic analysis revealed that serum IFN-{gamma} becomes detectable concomitantly with the release of mf at day 49 post-infection, peaks between day 77 and 98 post-infection (levels as in Table 2Go), and thereafter declines. Serum IFN-{gamma} in anti-IL-5-treated mice were found to have the same kinetics but at lower levels (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 2. Cytokine levels in the thoracic cavity and serum of L. sigmondontis-infected mice (pg/ml)a
 
For comparison, we also tried to measure NP-chemotactic cytokines in the thoracic cavity of non-infected mice, but none of these cytokines could be detected, neither in serum nor in the thoracic cavity (not shown).

In order to analyze the cellular source of NP-chemotactic cytokines in the thoracic cavity, thoracic cavity MP were purified. MP from infected control mice produced high amounts of TNF-{alpha}, in contrast to MP from anti-IL-5-treated mice (Table 3Go). Cells from both mice produced comparable amounts of IL-8, IL-12 and G-CSF, while those from treated mice produced more IL-10 (Table 3Go), arguing against a general deactivation with regard to cytokine production.


View this table:
[in this window]
[in a new window]
 
Table 3. Cytokine production by cultured thoracic cavity macrophages in response to LPS (1 µg/ml)a
 
Immunohistostaining with anti-TNF-{alpha}, anti-GM-CSF, anti-KC as well as anti-IL-4 antibodies was carried out using cytospins prepared from PLEC during microfilaremia. Cytospins from infected control mice revealed the formation of cell clusters consisting of EP and MP. Consistent with TNF-{alpha} production by MP isolated from infected control mice (Table 3Go), there was bright staining for TNF-{alpha} in MP, very prominently within clusters (Fig. 2GGo). In contrast, cytospins from anti-IL-5-treated mice showed that EP–MP clusters were absent and that MP did not stain for intracellular TNF-{alpha} (Fig. 2HGo). There was only low staining for GM-CSF by MP and IL-4 by EP in infected control mice (not shown), consistent with the relatively low levels in the thoracic cavity. Staining intensity of KC did not surpass background levels (not shown).

Production of NP-chemotactic TNF-{alpha} was also present in lung tissue, as evidenced by semiquantitative PCR (Table 4Go). There was a reduction (3-fold) in the levels of TNF-{alpha} mRNA in mice treated with anti-IL-5 compared to control mice. This difference was significant and prominent 80 days but not 28 days post-infection (Table 4Go).


View this table:
[in this window]
[in a new window]
 
Table 4. TNF-{alpha} mRNA levels in the lungs of infected BALB/c mice
 
Collectively these results demonstrate that depletion of IL-5 prevents the production of TNF-{alpha}, G-CSF and other NP-chemotactic cytokines (Tables 2 and 3GoGo), the formation of TNF-{alpha}-producing clusters at the site of infection (Fig. 2GGo) as well as production of TNF-{alpha} in the lungs (Table 4Go). The data further show that G-CSF which is reduced after anti-IL-5 treatment (Tables 1 and 2GoGo) is essential for nodule formation, and that TNF-{alpha} and other proinflammatory cytokines (Tables 2 and 3GoGo) are associated with NP accumulation in murine filariasis. These results suggest that NP-chemotactic cytokines are involved in IL-5-dependent NP recruitment.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The relevance of IL-5-dependent pathways in helminth infection is not well understood. IL-5 levels and EP counts are generally elevated in helminth infections (21) and there is evidence for killing of helminths by EP in vitro (e.g. 1,2,22). However, only in some experimental helminth infections does neutralization of IL-5 in vivo result in elevation of worm loads (3,4), whereas in others no effect is seen (6,7,23,24). Interestingly, participation of IL-5 in the formulation of granulomas was noted for murine Schistosoma japonicum infection (25). In filarial infection, EP but also NP were shown to be able to attack worms in vitro (26,27). Furthermore, histologic examination of worm-containing nodules of human and animal pathogenic Onchocerca species shows NP to form the innermost layer around live adult worms (28,29).

The data from this study suggest an IL-5-mediated mechanism of helminth control via NP-dependent nodule formation in vivo: NP are essential for nodule formation around adult filarial worms, as clearly demonstrated by NP depletion using anti-G-CSF (Table 1Go). This process leads to encapsulation of live worms (with a histologic pattern similar to nodules in human onchocerciasis) (28) and eventually results in worm killing. NP are dependent on IL-5 for normal migration to the site of infection and for formation of the inner layer of the inflammatory nodules (Table 1Go, and Figs 1 and 2GoGo). Since NP have been reported not to express the IL-5 receptor (20), there is possibly no direct effect of IL-5 on NP, e.g. mediating a survival signal as described for EP (20). Therefore, IL-5 apparently exerts its role on NP in an indirect manner, likely involving G-CSF, a cytokine which was shown in our model to be essential for the formation of inflammatory nodules and which is known to promote NP activation (30,31) as well as accumulation and local survival by apoptosis impairment (32). In addition, the mechanism of IL-5-mediated NP function probably involves TNF-{alpha}, an important mediator of granuloma formation in Schistosoma mansoni infection (33,34), and other NP-chemotactic cytokines (Fig. 2Go, and Tables 3 and 4GoGo).

The anti-IL-5 treatment regimen used in this study was not sufficient to completely eliminate EP and NP from the thoracic cavity. The remaining NP in the thoracic cavity of anti-IL-5-treated mice were apparently able to form some nodules which showed the same histologic pattern as nodules from infected control mice (not shown). These results underscore the importance of NP for the nodule-forming process, since reduction of NP by anti-IL-5 becomes manifest in a drastic reduction of nodules without primarily affecting the nodule architecture.

The pathway of IL-5 acting indirectly on NP migration and activation implies the participation of cells which are (i) responsive to IL-5 and (ii) producers of NP-chemotactic factors. These functions need not necessarily be exerted by the same cell types. EP, in our system, were found to migrate to the thoracic cavity earlier than NP, remaining there at high levels (Fig. 1AGo), and they were specifically targeted by anti-IL-5. However, of the NP-chemotactic cytokines described to be produced by EP (the majority of these being clearly documented only in human EP) (3541), none was found to be produced by EP as evidenced by intracellular staining on cytospins, except for some IL-4 (not shown) (42).

In contrast, cytospins showed a strong production of TNF-{alpha} by MP (Fig. 2GGo); this was corroborated by cell culture of thoracic cavity MP (Table 3Go). Given the fact the EP, in control infected mice, formed clusters with MP (Fig. 2GGo), it is possible that EP induced MP to produce NP-chemotactic cytokines, as has been discussed earlier (35). Interestingly, EP–MP cluster formation has been observed in other helminth infections (43) and the adherence of MP to filariae in vitro was shown to be enhanced in the presence of EP or EP-derived products (44,45). Part of the MP activation by EP may be due to engulfment of degenerated EP (46). Thus, MP activation in the presence of EP is a phenomenon already described several times. Regardless of their activation mechanism, MP at the site of infection are clearly a source of TNF-{alpha} production, while in the absence of IL-5 and EP, TNF-{alpha} production by MP is reduced 10-fold in vitro (Table 3Go). To the best of our knowledge, these findings demonstrate for the first time a relationship between the two cytokines IL-5 and TNF-{alpha} in an infection model.

Interestingly, MP were also producers of G-CSF in L. sigmodontis infection (Table 3Go). Although on a single-cell level, and after stimulation by LPS (Table 3Go), MP from anti-IL-5-treated and infected control mice did not produce significantly different amounts of G-CSF, the lower number of MP in anti-IL-5-treated mice (Fig. 1CGo, see also total cell numbers in the legend) can explain the significantly reduced levels of this cytokine in thoracic cavity fluids in the absence of IL-5 (Table 2Go). G-CSF was found essential for the formation of inflammatory nodules in L. sigmodontis infection (Table 1Go). Thus, it seems reasonable to assume that reduced G-CSF production by MP contributes to the diminished nodule formation in anti-IL-5-treated mice.

With regard to other NP-chemotactic factors, the lower MP numbers in anti-IL-5-treated mice may also account for the reduction in KC observed in thoracic cavity fluids of these mice (Fig. 1Go), although production of KC by MP in vitro was not statistically different (Table 3Go), similarly to G-CSF. Part of the reduction in the levels of TNF-{alpha} and KC in anti-IL-5-treated mice may also be attributable to the fewer NP, given that NP have been reported to be a source of these cytokines (47).

IFN-{gamma} is a cytokine which is usually not produced together with Th2 cytokines. However, a recent report showed that Candida-infected mice, in the absence of a Th2 response, are unable to mount a protective Th1 response after vaccination, possibly through the lack of Th1 induction by granulocytes (48). A similar sequence of events, albeit with blockade of IL-5, is assumed for L. sigmodontis infection, where a strong Th2 response also precedes IFN-{gamma} production: IFN-{gamma} is not measurable in the first 4–6 weeks post-infection, in contrast to a high Th2 response, but becomes detectable only from day 49 post-infection (9) (see also Table 2Go). Our data suggest that IFN-{gamma} production is dependent on a fully developed Th2 response.

The histological pattern of inflammatory nodules in L. sigmodontis resembles that of O. volvulus infection with NP forming the inner layer of cells around live worms (29). In addition, high IL-5 production in the hyper-reactive form of onchocerciasis (Sowda) (49) is associated with the formation of nodules with more inflammatory tissue around the worms, including NP (50). This study, by intervention in IL-5-dependent pathways, shows that in murine filariasis there is not only an association but a causal relationship between IL-5 and NP activity with regard to nodule formation.

In conclusion, this study demonstrates NP as effector cells essential for the formation of inflammatory nodules around filarial worms in vivo and reveals an IL-5-dependent mechanism for NP accumulation. It will be interesting to elucidate if there are single NP-chemotactic cytokines, other than G-CSF, under the control of IL-5 which are essential for this cascade or if there is so much redundancy in this pathway that only a blockade of many of these NP-chemotactic cytokines will affect nodule formation in murine filariasis.


    Acknowledgments
 
The expert technical assistance by Kerstin Nissen-Pähle and Ellen Strine is acknowledged. We are indebted to Dr J. Lee (Phoenix, AZ) for providing anti-MBP serum. The work formed part of a doctoral study of K. M. Al-Q. at the Faculty of Biology, University of Hamburg, Germany. This study received financial support from the Deutsche Forschungsgemeinschaft (grant Ho 2009/1-1 to A. H.), the Edna McConnell Clark Foundation (to A. H.) and the German Academic Exchange Service (DAAD to K. M. Al-Q).


    Abbreviations
 
EP eosinophil granulocyte
G-CSF granulocyte colony stimulating factor
GM-CSF granulocyte macrophage colony stimulating factor
LPS lipopolysaccharide
MBP major basic protein
MP macrophage
mf microfilariae
NP neutrophil granulocyte
PLEC pleural exudate cells
TNF tumor necrosis factor

    Notes
 
Transmitting editor: S. H. E. Kaufmann

Received 17 June 1999, accepted 23 February 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Butterworth, A. E., Sturrock, R. F., Houba, V., Mahmoud, A. A., Sher, A. and Rees, P. H. 1975. Eosinophils as mediators of antibody-dependent damage to schistosomula. Nature 256:727.[ISI][Medline]
  2. Greene, B. M., Taylor, H. R. and Aikawa, M. 1981. Cellular killing of microfilariae of Onchocerca volvulus: eosinophil and neutrophil-mediated immune serum-dependent destruction. J. Immunol. 127:1611.[Free Full Text]
  3. Korenaga, M., Hitoshi, Y., Yumaguchi, N., Sato, Y., Takatsu, K. and Tada, I. 1991. The role of IL-5 in protective immunity to Strongyloides venezuelensis infection in mice. Immunology 72:502.[ISI][Medline]
  4. Sasaki, O., Sugaya, H., Ishida, K. and Yoshimura, K. 1993. Ablation of eosinophils with anti-IL-5 antibody enhances the survival of intracranial worms of Angiostrongylus cantonensis in the mouse. Parasite Immunol. 15:349.[ISI][Medline]
  5. Lange, A. M., Yutanawiboonchai, W., Scott, P. and Abraham, D. 1994. IL-4- and IL-5-dependent protective immunity to Onchocerca volvulus infective larvae in BALB/cBYJ mice. J. Immunol. 153:205.[Abstract/Free Full Text]
  6. Sher, A., Coffman, R. L., Hieny, S. and Cheever, A. W. 1990. Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse. J. Immunol. 145:3911.[Abstract/Free Full Text]
  7. Finkelman, F. D., Shea-Donohue, T., Goldhill, J., Sullivan, C. A., Morris, S. C., Madden, K. B., Gause, W. C. and Urban, J. F., Jr. 1997. Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models. Annu. Rev. Immunol. 15:505.[ISI][Medline]
  8. Petit, G., Diagne, M., Marechal, P., Owen, D., Taylor, D. and Bain, O. 1992. Maturation of the filaria Litomosoides sigmodontis in BALB/c mice; comparative susceptibility of nine other inbred strains. Ann. Parasitol. Hum. Comp. 67:144.[ISI][Medline]
  9. Al-Qaoud, K. M., Taubert, A., Zahner, H., Fleischer, B. and Hoerauf, A. 1997. Infection of BALB/c mice with the filarial nematode Litomosoides sigmodontis: role of CD4+ T cells in controlling larval development. Infect. Immun. 65:2457.[Abstract]
  10. Al-Qaoud, K. M., Fleischer, B. and Hoerauf, A. 1998. The Xid defect imparts susceptibility to experimental murine filariosis—association with a lack of antibody and IL-10 production by B cells in response to phosphorylcholine. Int. Immunol. 10:17.[Abstract]
  11. Lopez, A. F., Strath, M. and Sanderson, C. J. 1984. Differentiation antigens on mouse eosinophils and neutrophils identified by monoclonal antibodies. Br. J. Haematol. 57:489.[ISI][Medline]
  12. Barsig, J., Bundschuh, D. S., Hartung, T., Bauhofer, A., Sauer, A. and Wendel, A. 1996. Control of fecal peritoneal infection in mice by colony-stimulating factors. J. Infect. Dis. 174:790.[ISI][Medline]
  13. Marechal, P., Le Goff, L., Petit, G., Diagne, M., Taylor, D. W. and Bain, O. 1996. The fate of the filaria Litomosoides sigmodontis in susceptible and naturally resistant mice. Parasite 3:25.[ISI][Medline]
  14. Hall, L. R., Mehlotra, R. K., Higgins, A. W., Haxhiu, M. A. and Pearlman, E. 1998. An essential role for interleukin-5 and eosinophils in helminth-induced airway hyperresponsiveness. Infect. Immun. 66:4425.[Abstract/Free Full Text]
  15. Folkard, S. G., Hogarth, P. J., Taylor, M. J. and Bianco, A. E. 1996. Eosinophils are the major effector cells of immunity to microfilariae in a mouse model of onchocerciasis. Parasitology 112:323.[ISI][Medline]
  16. Floyd, K., Suter, P. F. and Lutz, H. 1983. Granules of blood eosinophils are stained directly by anti-immunoglobulin fluorescein isothiocyanate conjugates. Am. J. Vet. Res. 44:2060.[ISI][Medline]
  17. Detlefs, R. L., Frieden, I. J., Berger, T. G. and Westrom, D. 1987. Eosinophil fluorescence: a cause of false positive slide tests for herpes simplex virus. Pediatr. Dermatol. 4:129.[Medline]
  18. Meyer Zum Buschenfelde, C., Cramer, S., Trumpfheller, C., Fleischer, B. and Frosch, S. 1997. Trypanosoma cruzi induces strong IL-12 and IL-18 gene expression in vivo: correlation with interferon-gamma (IFN-gamma) production. Clin. Exp. Immunol. 110:378.[ISI][Medline]
  19. Reiner, S. L., Zheng, S., Corry, D. B. and Locksley, R. M. 1993. Constructing polycompetitor cDNAs for quantitative PCR. J. Immunol. Methods 165:37.[ISI][Medline]
  20. Lopez, A. F., Elliott, M. J., Woodcock, J. and Vadas, M. A. 1992. GM-CSF, IL-3 and IL-5: cross-competition on human haemopoietic cells. Immunol. Today 13:495.[ISI][Medline]
  21. Finkelman, F. D., Pearce, E. J., Urban, J. F., Jr and Sher, A. 1991. Regulation and biological function of helminth-induced cytokine responses. Immunol. Today 12:A62.[Medline]
  22. Butterworth, A. E. 1984. Cell-mediated damage to helminths. Adv. Parasitol. 23:143.[ISI][Medline]
  23. Parsons, J. C., Coffman, R. L. and Grieve, R. B. 1993. Antibody to interleukin 5 prevents blood and tissue eosinophilia but not liver trapping in murine larval toxocariasis. Parasite Immunol. 15:501.[ISI][Medline]
  24. Kopf, M., Brombacher, F., Hodgkin, P. D., Ramsay, A. J., Milbourne, E. A., Dai, W. J., Ovington, K. S., Behm, C. A., Kohler, G., Young, I. G. and Matthaei, K. I. 1996. IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses. Immunity 4:15.[ISI][Medline]
  25. Cheever, A. W., Xu, Y. H., Sher, A. and Macedonia, J. G. 1991. Analysis of egg granuloma formation in Schistosoma japonicum-infected mice treated with antibodies to interleukin-5 and gamma interferon. Infect. Immun. 59:4071.[ISI][Medline]
  26. Aime, N., Haque, A., Bonnel, B., Torpier, G. and Capron, A. 1984. Neutrophil-mediated killing of Dipetalonema viteae microfilariae: simultaneous presence of IgE, IgG antibodies and complement is required. Immunology 51:585.[ISI][Medline]
  27. Johnson, E. H., Irvine, M., Kass, P. H., Browne, J., Abdullai, M., Prince, A. M. and Lustigman, S. 1994. Onchocerca volvulus: in vitro cytotoxic effects of human neutrophils and serum on third-stage larvae. Trop. Med. Parasitol. 45:331.[ISI][Medline]
  28. Wildenburg, G., Plenge-Bönig, A., Renz, A., Fischer, P. and Büttner, D. W. 1997. Distribution of mast cells and their correlation with inflammatory cells around Onchocerca gutturosa, O. tarsicola, O. ochengi, and O. flexuosa. Parasitol. Res. 83:109.[ISI][Medline]
  29. Rubio de Krömer, M. T., Krömer, M., Luersen, K. and Brattig, N. W. 1998. Detection of a chemotactic factor for neutrophils in extracts of female Onchocerca volvulus. Acta Trop. 71:45.[ISI][Medline]
  30. Lopez, A. F., Nicola, N. A., Burgess, A. W., Metcalf, D., Battye, F. L., Sewell, W. A. and Vadas, M. 1983. Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors. J. Immunol. 131:2983.[Abstract/Free Full Text]
  31. Demetri, G. D. and Griffin, J. D. 1991. Granulocyte colony-stimulating factor and its receptor. Blood 78:2791.[ISI][Medline]
  32. Ertel, W., Keel, M., Buergi, U., Hartung, T., Imhof, H. G. and Trentz, O. 1999. Granulocyte colony-stimulating factor inhibits neutrophil apoptosis at the local site after severe head and thoracic injury. J. Trauma 46:784.[ISI][Medline]
  33. Amiri, P., Locksley, R. M., Parslow, T. G., Sadick, M., Rector, E., Ritter, D. and McKerrow, J. H. 1992. Tumor necrosis factor alpha restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature 356:604.[ISI][Medline]
  34. Joseph, A. L. and Boros, D. L. 1993. Tumor necrosis factor plays a role in Schistosoma mansoni egg-induced granulomatous inflammation. J. Immunol. 151:5461.[Abstract/Free Full Text]
  35. Costa, J. J., Matossian, K., Resnick, M. B., Beil, W. J., Wong, D. T., Gordon, J. R., Dvorak, A. M., Weller, P. F. and Galli, S. J. 1993. Human eosinophils can express the cytokines tumor necrosis factor-alpha and macrophage inflammatory protein-1 alpha. J. Clin. Invest. 91:2673.[ISI][Medline]
  36. Sabin, E. A., Kopf, M. A. and Pearce, E. J. 1996. Schistosoma mansoni egg-induced early IL-4 production is dependent upon IL-5 and eosinophils. J. Exp. Med. 184:1871.[Abstract]
  37. Broide, D. H., Paine, M. M. and Firestein, G. S. 1992. Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics. J. Clin. Invest. 90:1414.[ISI][Medline]
  38. Braun, R. K., Franchini, M., Erard, F., Rihs, S., De Vries, I. J., Blaser, K., Hansel, T. T. and Walker, C. 1993. Human peripheral blood eosinophils produce and release interleukin-8 on stimulation with calcium ionophore. Eur. J. Immunol. 23:956.[ISI][Medline]
  39. Grewe, M., Czech, W., Morita, A., Werfel, T., Klammer, M., Kapp, A., Ruzicka, T., Schopf, E. and Krutmann, J. 1998. Human eosinophils produce biologically active IL-12: implications for control of T cell responses. J. Immunol. 161:415.[Abstract/Free Full Text]
  40. Girard, D., Paquin, R. and Beaulieu, A. D. 1997. Responsiveness of human neutrophils to interleukin-4: induction of cytoskeletal rearrangements, de novo protein synthesis and delay of apoptosis. Biochem. J. 325:147.[ISI][Medline]
  41. Woerly, G., Roger, N., Loiseau, S., Dombrowicz, D., Capron, A. and Capron, M. 1999. Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes. J. Exp. Med. 190:487.[Abstract/Free Full Text]
  42. Bober, L. A., Waters, T. A., Pugliese-Sivo, C. C., Sullivan, L. M., Narula, S. K. and Grace, M. J. 1995. IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils. Clin. Exp. Immunol. 99:129.[ISI][Medline]
  43. Walls, R. S., Hersey, P. and Quie, P. G. 1974. Macrophage–eosinophil interactions in the inflammatory response to Trichinella spiralis. Blood 44:131.[ISI][Medline]
  44. Haque, A., Ouaissi, A., Santoro, F., des Moutis, I. and Capron, A. 1982. Complement-mediated leukocyte adherence to infective larvae of Dipetalonema viteae (Filarioidea): requirement for eosinophils or eosinophil products in effecting macrophage adherence. J Immunol 129:2219.[Abstract/Free Full Text]
  45. Chandrashekar, R., Rao, U. R., Parab, P. B. and Subrahmanyam, D. 1986. Brugia malayi: rat cell interactions with infective larvae mediated by complement. Exp. Parasitol. 62:362.[ISI][Medline]
  46. Dvorak, A. M., Weller, P. F., Monahan-Earley, R. A., Letourneau, L. and Ackerman, S. J. 1990. Ultrastructural localization of Charcot–Leyden crystal protein (lysophospholipase) and peroxidase in macrophages, eosinophils, and extracellular matrix of the skin in the hypereosinophilic syndrome. Lab. Invest. 62:590.[ISI][Medline]
  47. Cassatella, M. A., Gasperini, S. and Russo, M. P. 1997. Cytokine expression and release by neutrophils. Ann. NY Acad. Sci. 832:233.[ISI][Medline]
  48. Mencacci, A., Del Sero, G., Cenci, E., d'Ostiani, C. F., Bacci, A., Montagnoli, C., Kopf, M. and Romani, L. 1998. Endogenous interleukin 4 is required for development of protective CD4+ T helper type 1 cell responses to Candida albicans. J. Exp. Med. 187:307.[Abstract/Free Full Text]
  49. Brattig, N., Nietz, C., Hounkpatin, S., Lucius, R., Seeber, F., Pichlmeier, U. and Pogonka, T. 1997. Differences in cytokine responses to Onchocerca volvulus extract and recombinant Ov33 and OvL3-1 proteins in exposed subjects with various parasitologic and clinical states. J. Infect. Dis. 176:838.[ISI][Medline]
  50. Korten, S., Wildenburg, G., Darge, K. and Buttner, D. W. 1998. Mast cells in onchocercomas from patients with hyperreactive onchocerciasis (sowda). Acta Trop. 70:217.[ISI][Medline]