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The Journal of Immunology, 2002, 168: 5199-5206.
Copyright © 2002 by The American Association of Immunologists

Expansion of NK Cells with Reduction of Their Inhibitory Ly-49A, Ly-49C, and Ly-49G2 Receptor-Expressing Subsets in a Murine Helminth Infection: Contribution to Parasite Control1

Simone Korten2,*, Lars Volkmann*, Michael Saeftel*, Kerstin Fischer*, Masaru Taniguchi{dagger}, Bernhard Fleischer* and Achim Hoerauf*

* Department of Medical Microbiology and Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; and {dagger} RIKEN Research Center for Allergy and Immunology, The Institute of Physical and Chemical Research, and Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Natural killer cell-associated direct cytotoxicity and cytokine production are crucial mechanisms for early innate host resistance against viruses, bacteria, or protozoa. The engagement of inhibitory NK cell receptors can influence host responses to viruses. However, these receptors have not been investigated to date in parasitic infections, and little is known about the role of NK cells in the defense against helminths. Therefore, we have correlated the frequencies of cells expressing the pan-NK marker DX5 and subsets bearing inhibitory Ly-49 receptors with worm survival and cytokine production during infection with Litomosoides sigmodontis in BALB/c mice (H2d), the only fully permissive model of filariasis. A marked influx of DX5+/CD3- NK cells and DX5+/CD3+ T cells into the pleural cavity, where the parasites were located, was observed. The frequency of pleural NK cells expressing the H2d-reactive inhibitory receptors Ly-49A, Ly-49C, or Ly-49G2 declined most strongly compared with spleen and blood. In the peripheral blood, longitudinal analysis revealed an early and stable reduction of Ly-49C+ and Ly-49G2+ NK cells, a subsequent significant increase of the entire NK cell and DX5+/CD3+ T cell populations, and a reduction in the Ly-49A+ subset. The in vivo depletion of NK cells strongly enhanced the worm load and influenced IL-4 and IL-5 plasma levels. These data demonstrate a new role for NK cells in the host defense against filariae and, for the first time, alterations of Ly-49 receptor-expressing NK cell subsets in a parasitic infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Helminthic infections affect more than one-tenth of the world’s population and cause considerable morbidity. For this reason, they demand the development of new treatment and vaccine strategies (1, 2). Understanding the fundamental immune mechanisms in the host-parasite relationship, including the role of NK cells, is an important prerequisite for this goal.

NK cells play an important role in the first line of defense against viral, bacterial, and protozoan infections (3, 4, 5), but few data exist on their role in immune responses against helminths. Increased NK cell activity has been reported for human trichinellosis (6), infection with Strongyloides, and chronic hyperreactive onchocerciasis (7). The only previous studies on experimental murine filariasis concern the influence of NK cells on adult worm development in Brugia malayi (8). We chose the natural infection of susceptible BALB/c mice by Litomosoides sigmodontis for investigating NK cells, because it is the only murine model in which filariae undergo a complete life cycle (9). This involves the transmission of infectious larvae by mites, the maturation of infectious larvae into adult worms within 28 days postinfection (p.i.,3 hereafter denoted as D28), prepatency (D28-D60), microfilaremia during patency (D60-D90), inflammatory nodule formation around the adult worms, and finally their elimination (D40-D120). In contrast with BALB/c, resistant C57BL/6 (B6) mice kill the adult worms in the prepatent phase.

An effective defense against L. sigmodontis in mice, as in human infection with Onchocerca volvulus (10, 11), is associated with both Th1- and Th2-driven immune responses involving IL-4, IL-5 enhanced Ab production, eosinophilia, and mastocytosis (9, 12, 13, 14, 15, 16), as well as IFN-{gamma}, macrophages, and neutrophils (17, 18, 19, 20).

Host immunity to the distinct stages of L. sigmodontis in BALB/c mice is complex. In ex vivo assays, filarial Ags induce both strong Th1- and Th2-type cytokines in the early prepatency. A temporary down-regulation of these cytokines, sparing IL-10, occurs toward the end of prepatency, followed by an increase of IFN-{gamma}, IL-4, and IL-13 during patency and postpatency (21). The persistence of microfilariae (mf) is facilitated by IL-10, but not by IL-4 or IFN-{gamma} (22). IL-5 is crucial for neutrophil-mediated worm encapsulation (14) and early protection against natural infection induced by irradiated infectious larvae (23). MHC haplotypes influence clearance of mf in susceptible, but not resistant strains (22). Irrespective of a host’s genetic background, the presence of just one adult female worm will skew immune responses to facilitate the persistence of mf (22). The role of NK cells during prepatency and patency is not known.

Due to the nonclonal nature of NK cells, they can respond more rapidly to infections than T lymphocytes and are the first lymphocytes recruited to sites of infection (24). They expand in blood, spleen, liver, and lung early in viral or protozoan infections and are potent producers of cytokines such as IFN-{gamma}, GM-CSF, IL-5, IL-10, and IL-13 (4, 24, 25, 26) that amplify innate immune responses and bias Ag-specific T cell responses (3, 24, 27). They also produce cytotoxic proteins such as perforins, granzymes, and serine proteases (28). This suggests that NK cells influence both early and late arms of the host immune response.

We hypothesized that NK cells could also play a role in chronic helminthic infections such as filariasis. Moreover, most filariae release likely NK cell modulators such as LPS-like molecules of their symbiotic endobacteria (Wolbachia spp.) and glycoproteins containing N-linked glycans (29, 30, 31, 32, 33, 34) in their excretory/secretory products. Additionally, they might kill mf directly via Fc{gamma}RIII receptors and Ab-dependent cellular cytotoxicity, as reported for schistosomal cercariae (35).

Intriguingly, direct killing and cytokine production by NK cells can be reduced by engagement of inhibitory receptors, which use intracellular immunoreceptor tyrosine-based inhibitory motifs to block signaling by activating receptors (36, 37). In mice, these lectin-like receptors of the Ly-49 or CD94/NKG2 family are well known to recognize MHC class I molecules on virus-infected cells, tumor cells, or bone marrow grafts (37). At present, 23 potential Ly-49 genes are known (Ly-49A-W), of which 13 are predicted to code for inhibitory (for example, Ly-49A, C, G2, I) and 10 for activating receptors (for example, Ly-49D, H) (38). These are expressed on partially overlapping NK cell subsets. Ly-49 surface expression levels and Ly-49+ NK cell subset frequencies can vary according to the presence of their cognate ligands, which are MHC class I molecules (39, 40) or carbohydrate moieties such as glycans (33, 41, 42, 43). Ly-49 receptors and other NK cell markers such as NK1.1 and the pan-NK marker DX5 can also be expressed by some conventional TCR{alpha}{beta} T CD8+ or CD4+ T cells as well as by non-MHC-restricted {gamma}{delta} T cells and CD1d-restricted NKT cells (44, 45, 46, 47, 48). The latter are T lymphocytes with intermediate TCR/CD3{epsilon} and restricted V{alpha}{beta} chain expression (85% V{alpha}14J{alpha}281 and 50% V{beta}8.2 chains). The up-regulation of NK cell-associated molecules on CD8+ T cells confers an additional capacity for non-MHC-restricted killing (46). Ly-49 inhibitory receptor expression can modify T cell responses; moreover, by blocking TCR signaling and reducing NK cell responses, it can exacerbate viral infections (47, 48, 49, 50, 51, 52, 53). Thus, the expression of inhibitory receptors in an infection may prove harmful to the host. To date, no data exist on its role during parasitic infections.

For the first time, we report on variations of NK cell subsets with inhibitory receptors at the sites of parasitic infection using a murine model of human filariasis. Furthermore, we correlate expansions of the entire populations both of NK cells and of T cells with NK markers with a reduced worm survival and altered cytokine expression, highlighting an overall contribution of NK cell-associated functions to the control of helminthic infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and infection with L. sigmodontis

Normal BALB/c mice were bred at the animal facilities of the Bernhard Nocht Institute (originally derived from Charles River Breeding Laboratories, Sulzfeld, Germany) and kept under specific pathogen-free conditions. Regular tests excluded any viral, bacterial, or other parasitic infections. Natural infections of mice with L. sigmodontis were performed using infectious mites, as described previously (9). NK cell-depleted and control BALB/c were investigated during the natural course of infection. A total of 50% of each group was sacrificed on D63, and the other 50% on D84 for the studying of pleural and splenic NK cells in the late phase of infection. Noninfected controls were three groups of five naive mice each. As additional controls, we infected J{alpha}281-/- BALB/c mice, which lack V{alpha}14 NKT cells (54), in two separate experiments of five mice each along with wild-type BALB/c mice as controls.

In vivo depletion of NK cells or CD8+ T cells

As BALB/c mice do not express NK1.1, they were depleted of NK cells by injecting rabbit antiasialo-GM1 antiserum i.p. (25 µl/mouse, 1/8 diluted with 0.5x PBS; WAKO, Richmond, VA) every 5 days from D35 to D84. The experiment was performed twice with control mice given PBS only. NK cell depletion was confirmed by flow cytometry analysis (FACScan; BD Biosciences, Heidelberg, Germany). During the course of the experiments, no mice succumbed to any disease. In two additional experiments, CD8+ T cells were depleted in three or six mice, by administering purified anti-CD8 mAb in a same schedule as above (the Ab-producing hybridoma YTS 169.4 was kindly provided by H. Waldmann, Oxford, U.K.). CD8+ T cell depletion effect was confirmed by FACS.

Quantification of parasites and inflammatory nodules

To quantify adult worms, inflammatory nodules, and pleural exudate cells, the thoracic cavity, in which the majority of adult worms reside (9), was flushed with two successive 2 ml PBS samples. The supernatant from the first was used for the detection of cytokines. Adult worms, mf, nodules, and cells were pooled from both washes and counted. Mf were also counted in 50 µl EDTA-treated blood after staining with Hinkelmann’s solution (0.5% w/v eosin Y, 0.5% w/v phenol, and 0.185% v/v formaldehyde in distilled water), as described previously (9). Microfilaremia was measured weekly between D55 and D84.

Abs and reagents for flow cytometry

We used FITC- or PE-conjugated mAbs, including anti-Ly-49A+D (12A8), anti-Ly-49C/I (5E6), and Ly-49G2 (LGL-1) against inhibitory Ly-49 receptors, as well as the pan-NK cell marker anti-DX5 PE (DX5) and anti-CD3{epsilon} FITC (145-2C11) to label T cells, from BD PharMingen. Because Ly-49I is not expressed by BALB/c mice, mAb 5E6 identifies Ly-49C only (55). Similarly, mAb 12A8 detects only Ly-49A and not Ly-49D in BALB/c mice. Anti-CD4 FITC (YTS 191.1), anti-CD8{alpha} PE (YTS 169.4), anti-B220 PE (CD45R Ly-5 Pan B cell, RA3-6B2), and anti-CD11b FITC (Mac-1 {alpha}-chain, M1/70.15) were purchased from Medac (Hamburg, Germany). Isotype control mouse IgG mAbs (BD PharMingen) were used as negative staining controls. To block nonspecific binding to Fc{gamma}RII/III, an anti-Fc{gamma}RII/III mAb (BD, 2.4G2) was used at 1:10.

Lymphocyte separation, flow cytometry analysis, and counting

Approximately 250 µl blood was taken weekly from the retro-orbital plexus in EDTA-coated capillary tubes from D35 to D84 in the NK cell-depleted mice, and in the control mice also, from the first week before infection. Blood (80 µl) was centrifuged to obtain 30 µl plasma. After hypotonic lysis of RBC from the remaining {approx}170 µl and washing with PBS/1% BSA, nucleated cells were adjusted to 1 ml from each mouse. A total of 100-µl aliquots was first blocked in V-bottom microwells with 2.4G2 mAb for 10 min before incubating for 0.5 h with the respective primary Abs, washing with PBS/1%BSA, fixing in 1% formaldehyde, and analyzing by FACS. Forward and side scatter were used to gate on the lymphocyte population, and 10,000 gated events were collected for analysis by CellQuest software. Pleural exudate cells were adjusted to 2 x 106/ml (in PBS/1% BSA) and 100 µl stained with mAbs, as described above. Spleen cell suspensions were prepared in PBS/1% BSA, followed by hypotonic lysis and washing, and adjusted to 2 x 106/ml, before staining, as described above.

Absolute lymphocyte subset numbers for spleen and pleural exudate cell suspensions were determined from the total cell count (Neubauer’s counting chamber) and FACS analysis, for blood, from PBMC counted as above, an estimated average total volume of 2.5 ml, and FACS analysis.

Cytokine assays

Cytokine concentrations (IFN-{gamma}, IL-4, IL-5, and IL-10) were determined in supernatants of the pleural exudates (thoracic wash) on D63 and D84 as well as in weekly serum samples from D42. In the first experiment, individual sera were diluted 1/10 with PBS/1% BSA; in the second, they were pooled from two mice each and diluted 1/5 to maximize sensitivity. The cytokine concentrations were measured with standard ELISAs: the Ab pairs for capture and detection (biotinylated) were purchased from BD PharMingen in the combinations recommended. Recombinant cytokines (BD PharMingen and R&D Systems, Wiesbaden, Germany) were used as standard positive controls according to the manufacturer’s instructions. All ELISAs were developed after incubation with streptavidin-peroxidase complex (1:10,000; Boehringer Mannheim, Mannheim, Germany), using 3,5,3',5' tetramethylbenzidine as substrate (Roth, Karlsruhe, Germany; dissolved 6 mg/ml in DMSO); sensitivities were 20 pg/ml for all cytokines.

Statistical analysis

Analysis of data was performed with Statview (version 5.0, Macintosh software; SAS Institute, Cary, NC) and Excel (Microsoft Excel 98 software, Macintosh edition; Redmond, WA). Significances were tested using: 1) for normally distributed parameters (blood), the paired Student t test for kinetics in the same mice and the unpaired Student t test to compare between depleted and control groups; 2) for non-normally distributed parameters (spleen and pleural cavity), the Mann-Whitney U test to compare compartments or parasite loads of different mice between days or between depleted and control groups; and 3) the Wilcoxon signed rank test for paired comparisons between the compartments of the same mice. An index was formed of the number of inflammatory nodules per live adult worm, and means were compared by Mann-Whitney U test. Differences were considered significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increase of NK cells and DX5+/CD3high/int T cell frequencies during infection

Both NK cells and a subset of T cells express the pan-NK marker DX5, but NK cells do not express CD3{epsilon}. Therefore, NK cells (DX5+/CD3-) and T cells (DX5+/CD3+) were distinguished by double staining and monitored in the blood of infected BALB/c mice. As shown in Fig. 1Go, A and C, and Fig. 2GoA, there was a significantly increased proportion of NK cells within the lymphocyte population between D35 and D84 above preinfection levels (D0). Absolute numbers of NK cells also increased substantially (Table IGo). Furthermore, more NK cells expressed DX5 weakly (DX5int) on D63 ({approx}30%) than on D0 ({approx}20%, Fig. 1GoC), and the population of DX5high NK cells was also enhanced. In addition, T cells with intermediate expression of DX5 and high or intermediate expression of CD3 (CD3high/int, hereafter denoted as CD3+) rose too, but less than did the NK cells (Fig. 1Go, B and C, and Fig. 2GoB; Table IGo). In both experiments, the NK cells and DX5+/CD3+ T cells showed biphasic increases after D28, with similar peaks around D40 and D63 (Fig. 1Go, A and B).



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  		FIGURE 1. Rising frequencies of DX5+/CD3- NK cells (A) and DX5+/CD3high/int T cells (B) in the blood of BALB/c mice during the natural course of infection. C, In representative dot plots, we use a cutoff value of 100 to assess the increase of DX5high and DX5int NK cell subsets (upper left quadrant) on D63 as well as for a CD3high/int expression on T cells. Data are expressed as the mean ± SE of 18 mice/group until D63 and of 9 mice/group thereafter. Each week, the p.i. frequencies were compared with the preinfection frequency (D0). Significant differences (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 by paired Student’s t test) were observed from D35 on (arrow). Results were similar in a third experiment (five mice).

 


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  		FIGURE 2. Increase in NK cell (A) and DX5+/CD3high/int T cell (B) frequencies in blood and pleural cavity vs spleen between D0, D63, and D84 p.i. C, Representative dot plots from D84 with same regions for DX5high and DX5int as in Fig. 1Go. Means ± SE were calculated together from 15 naive mice for D0 as well as 3 and 6 mice from two experiments for D63 and D84 each. Results were similar in a third experiment with 5 mice for D63 and D84 each (*, p < 0.05; **, p < 0.01; ***, p < 0.001 by Wilcoxon signed rank test comparing compartments on the same day, asterisks only; by Mann-Whitney U test for comparing spleen or pleural cavities; and by paired Student’s t test for comparing PBL between time points, asterisks on brackets).

 

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  		Table I. Absolute NK and T cell counts (x106) (±SD) in uninfected BALB/c mice (D0) and during infection (D63 and D84)1

 
We found no concomitant decreases in other T cell populations. The relative DX5-/CD3+, CD4+, and CD8+ T cell proportions remained largely unaltered during the course of infection (Fig. 3Go, B–D); their absolute numbers increased, although less sharply than the NK cells did (Table IGo).



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  		FIGURE 3. Sustained depletion of blood and pleural DX5+/CD3- NK cells and a minor DX5+/CD3+ T cell subset after five daily injections of antiasialo-GM1 antiserum from D35 to D84 p.i. A, Representative dot plots from PBL on D36 p.i. plus kinetics of NK cell and T cell frequencies (means ± SE) in blood, spleen, and pleural cavity postdepletion relative to those before depletion for PBL on D34, or on D0 for spleen and pleural cavity. B, Unchanged frequencies of DX5-/CD3+ T cells; C, CD8+ T cells; and D, CD4+ T cells. The data were obtained during the same depletion experiments as in Fig. 6Go and Table IIGo (*, p < 0.05 by unpaired Student’s t test for comparing depleted and control PBL and by Mann-Whitney U test for comparing spleen or pleural cavity between depleted and control mice).

 
Next, we tested for parallel expansions of NK cells and DX5+/CD3+ T cells in the pleural cavity and the spleen. Spleens, where NK cells are normally more numerous and frequent, showed higher frequencies than in blood and pleural cavities on D0 (Fig. 2GoA, Table IGo). Infected mice regularly showed a splenomegaly, but with significant decreases of NK cell proportions (Fig. 2GoA) and absolute counts (Table IGo) until D84. About 40% of NK cells were DX5int on D84 as in the pleural cavity, whereas these were only 20% in the blood (Fig. 2GoC). Similar to NK cells, relative frequencies (Fig. 2GoB) and absolute counts (Table IGo) of splenic DX5+/CD3+ T cells were higher than those of blood and pleural cavities on D0. Although their relative frequencies did not change (Fig. 2GoB), absolute numbers were temporarily decreased (Table IGo). Splenic DX5-/CD3+ T cell numbers were slightly lower by D63 (Table IGo), but their relative frequencies remained unaltered (Fig. 3GoB).

As with blood (although not spleen), the pleural cavities of infected mice contained substantially increased numbers and proportions of NK cells (Table IGo, Fig. 2GoA). In this study, in particular, distinct DX5int and DX5high NK cells were found (Fig. 2GoC). In addition, the DX5+/CD3+ T cell population had increased by D63 (Fig. 2GoB; Table IGo). Other T cell subsets also accumulated in the pleural cavity, particularly around D63 (Table IGo; Fig. 3Go, B–D).

In summary, NK cells and DX5+/CD3+ T cells, in particular, expanded with infection, especially in the blood and the pleural cavity, where the parasites were mostly concentrated.

Decreases of inhibitory Ly-49A, Ly-49C, and Ly-49G2 receptor-expressing NK cells in blood during infection

In the blood of uninfected BALB/c mice, {approx}50% of all DX5+ NK cells expressed the inhibitory Ly-49C receptor, {approx}30% Ly-49G2 (Figs. 4Go and 5GoB), and {approx}5% Ly-49A (Fig. 5GoC). The Ly-49C+ and Ly-49G2+ subsets declined in the first 4 wk p.i. (whereas total NK cell and DX5+ T cell frequencies increased later), reaching their lowest levels around D56 (Fig. 4GoA), and remaining significantly reduced through to D84. Interestingly, very few of the DX5int NK cells coexpressed Ly-49C or Ly-49G2, and the DX5-/Ly-49C+ population was also lower on D63 than D0 (Fig. 4GoB). By contrast, the Ly-49A+ NK cells were reduced later in the blood (between D63 and D84), but not in the other two compartments on D84 (Fig. 5GoC).



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  		FIGURE 4. Decreasing Ly-49C+ and Ly-49G2+ NK cell subset frequencies in the blood p.i. A, Kinetics and comparison of each p.i. time point with D0 frequencies of uninfected mice (p < 0.001–0.05 by paired Student’s t test for both subsets). B, Representative dot plots of anti-DX5- and anti-Ly-49C-stained PBL on D63 compared with D0. Mean ± SE of 18 mice/group until D63 and of 9 mice/group thereafter. Results were similar in a third experiment (5 mice).

 


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  		FIGURE 5. Comparison of Ly-49C+ (A), Ly-49G2+ (B), and Ly-49A+ (C) DX5+ NK cell frequencies in the blood, spleen, and pleural cavity between D0, D63, and D84 p.i. B, Ly-49G2+ NK cells, and C, Ly-49A+ NK cells. Means ± SE for A and B were calculated from 15 naive mice for D0, also from 3 and 6 mice from two experiments for D63 and D84 each. Results were similar in a third experiment (5 mice). For C, means were calculated from 15 naive mice for D0 and one experiment with 5 mice each for D63 and D84 (*, p < 0.05, and **, p < 0.01 by the same statistical tests as in Fig. 2Go).

 
Decreases of Ly-49A+, Ly-49C+, and Ly-49G2+ NK cells in the pleural cavity

Frequencies of Ly-49C+ and Ly-49G2+ pleural NK cells were lower than in spleen and blood on D0 and still similar by D63, but they were particularly strongly reduced on D84, by when they had returned to normal in the spleens (Fig. 5Go, A and B). Pleural Ly-49A+ NK cells showed a temporary reduction and the splenic population an increase on D63 (Fig. 5GoC).

In summary, DX5+ cells bearing the inhibitory Ly-49A, Ly-49C, or Ly-49G2 receptor were significantly reduced during infection, especially in the pleural cavity, where the Ly-49C+ and Ly-49G2+ subsets were reduced even more than in the blood.

Depletion of NK cells reduced the elimination of adult worms and mf

Next, we tested whether the expansion of NK cells during the late phase of infection affected parasite loads. Therefore, we depleted NK cells by injecting antiasialo-GM1 antiserum, which strongly and significantly reduced NK cells in the blood, spleen, and especially the pleura (Fig. 3GoA). Absolute NK cell numbers were lowered, too, despite similar total pleural lymphocyte counts (control vs depleted: 3.2 ± 0.9 x 106 vs 0.7 ± 0.9 x 106 and 1.1 ± 0.1 x 106 vs 0.2 ± 0.2 x 106, respectively). DX5+/CD3+ T cells were also reduced in blood and pleural cavity, but not in the spleen (Fig. 3GoA). By contrast, the majority of T cells and their subsets was largely unaffected in blood, spleen, and pleural cavity (Fig. 3Go, A–D) and absolute numbers even higher in the pleural cavity on D84 (756,000 ± 202,500 vs 360,640 ± 215,740).

In untreated mice, the elimination of the adult worms progressed between D63 and D84, as evidenced by fewer live mf and adult worms without host cell aggregations and more inflammatory nodules frequently containing dead worms (Table IIGo).


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  		Table II. Effects of NK cell depletion on the worm burden in vivo on D63 and D84 postinfection1

 
Although the depletion of NK cells did not influence the number of inflammatory nodules, the load of live adult worms was 2- to 3-fold higher on both D63 and D84 (Table IIGo). Furthermore, the total number of mf was greatly enhanced on both days in the pleural cavity and significantly on D63 in the blood (Table IIGo).

Therefore, NK cells apparently contributed to the elimination of macrofilariae and mf because their depletion resulted in a significantly higher number of live worms.

Deficiency of V{alpha}14 NKT cells or depletion of CD8+ cells had no influence on the worm burden

To test whether CD1d-restricted V{alpha}14 NKT cells were involved, we infected J{alpha}281-/- mice, which lack V{alpha}14 NKT cells. They showed no significant increases in numbers of adult worms, mf, or nodules above those in wild-type BALB/c mice (Table IIIGo). Depletion with an anti-CD8 mAb influenced neither the pleural nor the peripheral microfilarial load (depleted vs control mice on D60, 12 ± 11 vs 7 ± 12; D80, 2 ± 2 vs 3 ± 5, p > 0.05) nor the adult worm load (D60, 18 ± 18 vs 19 ± 4; D80, 6 ± 5 vs 3 ± 4, p > 0.05).


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  		Table III. The worm burden was not affected in J{alpha}281-/- (V{alpha}14 NKT-deficient) mice1

 
The depletion of NK cells affected cytokine production

We further assessed contributions of NK cells in the late phase of infection by testing the effect of their depletion on levels of IFN-{gamma}, IL-4, IL-5, and IL-10 in pleural exudates and weekly plasma samples. The plasma levels of IFN-{gamma} increased steadily (in two experiments) in both depleted and control mice between D70 and D84 from initially very low levels (Fig. 6Go). Results with the other cytokines varied in the two experiments. No significant differences were observed with depletion in the first experiment. Interestingly, in the second, IL-4 and IL-5 levels increased only in the depleted, but not the control mice between D70 and D84 (p = 0.0495); with IL-10, they were very high, but so variable that the increases did not achieve significance (Fig. 6Go). IL-4, IL-5, and IL-10 plasma levels were generally low in control mice in both experiments. Pleural IFN-{gamma}, IL-4, IL-5, or IL-10 levels did not differ between depleted and control mice (data not shown).



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  		FIGURE 6. Late effect of NK cell depletion on plasma cytokine levels. Cytokine concentrations were determined by ELISA (means ± SE; *, p < 0.05 by Mann-Whitney U test). To increase sensitivity, samples from the second experiment were pooled from pairs of the 12 mice/group until D63 and the 6 mice/group thereafter. The late increase of IFN-{gamma} was also observed in the first experiment, but not that observed in the other cytokines (nonpooled samples from 6 mice/group until D63 and 3 mice/group until D84; data not shown).

 
In summary, the depletion of NK cells sometimes enhanced plasma levels of IL-4, IL-5, and IL-10, but not of IFN-{gamma}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we analyzed for the first time frequencies of NK cells and their expression of inhibitory receptors during the course of a helminth infection, and tested their functional relevance during patency. We focused on three compartments: 1) the peripheral blood, as an indicator of NK cell mobilization and of microfilaremia; 2) the spleen as NK cell-rich organ; and 3) the pleural cavity, the main site of inflammatory defense against the adult worms and freshly released mf. During the natural course of infection, NK cells and DX5+/CD3+ T cells increased greatly in frequency and absolute numbers in the pleural cavity and blood, but they decreased in the spleens, in contrast with protozoan infections (56). Thus, the NK cell response was focused on the main infected sites, suggesting redistribution from the spleen. Remarkably, both NK and DX5+/CD3+ T cells showed biphasic increases with peaks around D40 and D63, suggesting that the response was provoked not by the infectious larvae, but by the pleural location of the adult worms and release of mf.

NK cells can be recruited directly by pathogens or tumor cells (57), but they are attracted into infected tissues primarily by cytokines or chemokines (58). In peripheral tissues, NK cell populations can expand further in an autocrine manner (59, 60). The increase in DX5int NK cells with little Ly-49 expression may indicate such a proliferating population, according with a recent report (61). LPS stimulates the proliferation of NK cells, a process that depends on the presence of APCs or T cells (34). Several factors might drive NK cells to accumulate around L. sigmodontis, such as cytokines induced by parasite-derived glycans and bacterial LPS-like molecules from Wolbachia endosymbionts in filariae (30, 31). Such LPS-like molecules are known to induce macrophages to produce TNF-{alpha} (30, 31), which could activate NK cells (6), as well as attracting neutrophils (62). Worm-derived glycans induce IL-4, IL-5, IL-10, and eosinophilia (63), and may influence cytokine production by NK cells (see below). Endosymbiont DNA from decaying bacteria could activate NK cells to produce cytokines and become more cytotoxic, as shown for eubacterial DNA (64).

Interestingly, we found that DX5+/CD3+ T cells also accumulated in the pleural cavity and peripheral blood during infection. The DX5 Ag, an {alpha}2 integrin, is expressed by activated CD4+ or CD8+ {alpha}{beta} T cells (46, 51, 61) and {gamma}{delta} T cells, which can be either CD4+, CD8+, or double negative (DN) (65). The observation that pleural CD4+ and CD8+ T cells rose, but then decreased, whereas the increase in DX5+/CD3+ cells was more stable, may implicate DN {gamma}{delta} T cells. These are also involved in human infection with O. volvulus (66). CD3int T cells might represent activated conventional T cells with reduced TCR levels rather than the V{alpha}14 NKT cells, whose deficiency clearly did not affect the worm load.

In infected mice, we demonstrate a highly significant reduction in peripheral Ly-49C+ and Ly-49G2+ DX5+ cells, which represent the two main H-2d-reactive inhibitory NK cell subsets in the BALB/c strain. This reduction was even more pronounced at the most heavily infected site, the pleural cavity. Moreover, because it was most conspicuous on the mature DX5+ population, it does not merely reflect dilution by newly generated DX5int NK cells with weak or negative Ly-49 expression (67). Indeed, Ly-49C+ and Ly49-G2+ subsets fell in the blood before total NK cells increased. The Ly-49G2+ and Ly-49A+ subsets rose only in the spleen. In line with other results, Ly-49A was expressed on a rarer subset than the other two receptors (68) and showed minor reductions. Hence, these results provide the first sign of variation in expression of inhibitory Ly-49 receptors on mature NK cells locally and systemically during a parasitic infection. This might be influenced by factors released or evoked by the parasites, perhaps including cytokines and yet unknown changes in surface or soluble H2d MHC class I expression. In addition to worm-derived peptides and glycans (41, 42, 69, 70, 71), these factors could modulate expression of the H2d-reactive Ly-49A, Ly-49C, and Ly-49G2 receptors, as suggested by other studies (52, 72, 73).

According to the more common notion, Ly-49+ subset frequencies and Ly-49 surface expression levels are down-regulated during maturation by new, cognate MHC class I ligands in the host environment (39, 40). However, mature NK cells also modulate their Ly-49 expression according to the in vivo MHC class I environment after transfer (73) and at the site of viral infection (52). In conclusion, we suggest that this observed reduction of subsets bearing inhibitory receptors might enhance NK cell-mediated defense, in particular of DX5high NK cells. They may represent a more mature and cytotoxic population than DX5int NK cells (61). A potential cytotoxic function of NK cells is also suggested by the direct adherence of pleural exudate cells from control, but not from NK cell-depleted C57BL/6 mice to adult worms, a preliminary observation that we are now pursuing.

A contribution of NK cells to parasite containment is corroborated by our observation that depletion with antiasialo-GM1 antiserum resulted in a significantly higher number of live adult worms and mf. Depletion had little effect, if any, on DX5-/CD3+, CD4+, and CD8+ T cells or macrophages (not shown); therefore, the majority of T cells did not express the asialo-GM1 Ag in contrast to a murine systemic viral infection (51). However, it did reduce DX5+ T cells, possibly by removing few asialo-GM1-expressing DN {gamma}{delta} T cells (74), because depletion with an anti-CD8 mAb had no effect.

Surprisingly, plasma IFN-{gamma} rose equally in depleted and control mice, while in one experiment, IL-4 and IL-5 levels increased after depletion between D70 and D84, being low in control mice, as described before (16, 21). Microfilaremia induced higher IL-5 than IL-4 levels in both groups, as we stated previously (16). Although both cytokines are necessary for protection (14, 16), whereas IL-10 seems to suppress it (22), the enhanced levels of all three cytokines after depletion were linked with a higher burden of worms, possibly reflecting the aggravated disease they caused. However, in this model, cytokine levels did not correlate with the parasitic load; both in depleted and control mice, the number of worms declined between D63 and D84, just when the cytokine levels were rising. Therefore, we propose that the cytokine balance was influenced by NK cells and perhaps some DX5+/CD3+ T cells rather than the parasites alone. Although no evidence for a true Th1-Th2 shift was obtained, we hypothesize that NK cells maintain a defense-promoting milieu. In human and experimental filarial infections, it is the balanced cooperation of Th1- and Th2-type responses, rather than any polarizing shift, that leads to the most favorable outcome (1). As our depletion results argue against direct IFN-{gamma}, IL-5, or IL-10 production by NK cells, we suggest that they produce other cytokines such as TNF-{alpha}, GM-CSF, or IL-8, which promote cellular responses. These latter cytokines, together with IFN-{gamma}, are crucial for nodule formation, especially by activating neutrophils (16, 18, 19, 62).

In conclusion, the novel decrease in Ly-49 receptor expression and subsequent expansion of the total NK cell population, especially where the parasites are most concentrated, plus their further multiplication after NK depletion, together provide strong evidence of NK cell-mediated defense against helminths. These data support the current hypothesis that Ly-49 receptor expression influences defense mechanisms during infections using the murine infection with L. sigmodontis as a natural model for studying NK cell biology.


   Acknowledgments
 
We thank Martin Mempel for useful comments and Christian Bogdan for critical reading of the manuscript.


   Footnotes
 
1 This study was supported by the German Research Foundation (Grants Ho/2009-1/1, Ho/2009-1/2, and Ho/2009-1/3). Back

2 Address correspondence and reprint requests to Dr. Simone Korten at the current address: Molecular Immunology Group, Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital (JR2 Level 7), Oxford OX3 9DU, U.K. E-mail address: simone.korten{at}ndm.ox.ac.uk Back

3 Abbreviations used in this paper: p.i., postinfection; DN, double negative; int, intermediate; mf, microfilariae; D, day. Back

Received for publication May 3, 2001. Accepted for publication March 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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