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Departments of Medicine and Immunology, Baylor College of Medicine
Department of Pathology, University of Texas Health Science Center, Houston, Texas
Equipe Avenir, INSERM U, Hopital St-Vincent de Paul, Paris
Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidium-infected epithelial cells in a dose-dependent manner. Lysis was due to CD3-CD16+CD56+ cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16Hi cytolytically active cells. Major histocompatibility complex class Irelated molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cellmediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.
Parasites of the genus Cryptosporidium are coccidian protozoans that infect intestinal epithelial cells, causing diarrhea. The mucosal defenses involved in clearance of human infection with this parasite are poorly understood. CD4+ T cells and interferon (IFN) play pivotal roles in recovery from murine cryptosporidiosis [14]. IFN- is produced by stimulated cells from patients who have recovered from cryptosporidiosis [5] and in intestinal tissue of sensitized individuals [6], but IFN- is not expressed during primary Cryptosporidium infection [68]. Thus, IFN-independent pathways appear important in human infection. In testing for IFN-independent pathways, we identified interleukin (IL)15 in the jejunum after experimental Cryptosporidium infection [9]. Volunteers expressing IL-15 shed fewer oocysts than did seronegative volunteers not expressing this cytokine.
IL-15 functions include regulation of cytokine expression by antigen-presenting cells and chemokine production by intestinal epithelial cells [10, 11]. In addition, IL-15 increases cytotoxic activity of NK and CD8+ T cells [12, 13]. IL-15 treatment of intraepithelial lymphocytes leads to lysis of intestinal epithelial cells [1416]. On the basis of these data, we hypothesized that IL-15mediated activation of NK cells may be critical for resolving human cryptosporidiosis.
SUBJECTS, MATERIALS, AND METHODS
Peripheral blood mononuclear cell (PBMC) isolation/enrichment and culture.
Blood samples were collected from 7 consenting healthy volunteers, as approved by the Institutional Review Board of the Baylor College of Medicine. Serum from the volunteers was tested for Cryptosporidium antibodies by use of a standardized ELISA [17]. Results of this assay correlate with resistance to C. parvum challenge and intestinal IFN- expression and correlate inversely with IL-15 production [9]. PBMCs were separated from heparinized blood by density-gradient centrifugation using Histopaque-1077 (Sigma Diagnostics). In some experiments, isolated PBMCs were depleted of CD3+ T cells or CD16+CD56+ NK cells by magnetic cell sorting using purified mouse antihuman CD3, CD16, and CD56 antibodies (Pharmingen) alone or in combination (1 g/1 × 106 target cells; 30 min at 25°C), by use of antimouse pan-IgGcoated magnetic beads (Dynal Biotech) and a magnetic particle concentrator (Dynal Biotech). NK cells were isolated by negative selection using a commercial kit (Dynal Biotech). The isolated NK cell population was >90% pure, as assessed by flow cytometry.
PBMCs (the whole population or depleted populations) were cultured (2 × 106 cells/mL) in RPMI 1640 with 2 mmol/L L-glutamine and supplemented with 10% heat-inactivated fetal bovine serum (FBS), 15 mmol/L HEPES, 100,000 U/L penicillin, 100,000 g/L streptomycin, and 250 g/L amphotericin B (RPMI-c) with or without human recombinant IL-15 (rIL-15) (0, 1, or 10 ng/mL; Chemicon). NK cells were cultured in 24-well plates (1 × 106 cells/mL) in complete medium containing different concentrations of rIL-15 (up to 72 h at 37°C in 5% CO2).
In vitro and ex vivo cultures.
Human colon carcinoma cell line HCT-8 (CCL-244; American Type Culture Collection ) cells were cultured in RPMI-c supplemented with glutamine. A second line, Caco-2 (HTB-37; ATCC), was grown in Dulbecco's MEM (DMEM) containing 2 mmol/L L-glutamine and 4.5 g/L D-glucose with 10% heat-inactivated FBS, 0.1 mmol/L MEM nonessential amino acids, 15 mmol/L HEPES, 100,000 U/L penicillin, 100,000 g/L streptomycin, and 250 g/L amphotericin B.
Ileal tissue was transported to the laboratory in CMRL Medium-1066 (Invitrogen). The mucosal layers were removed, and the tissue was sectioned into 3-mm2 pieces and placed lumen-side up onto collagen-treated membranes (0.4-m pore size) of transwell insert cups (Costar). The inserts were placed into wells containing CMRL Medium-1066 supplemented with 5 g/L D-glucose, 0.3 mmol/L Glutamax (Invitrogen), 0.3 g/L Tricine (Fisher Scientific), 1 mg/L L-methionine (Fisher Scientific), 0.55 mg/L hydrocortisone (BD Biosciences), 5% heat-inactivated FBS, 100,000 U/L penicillin, 100,000 g/L streptomycin, and 250 g/L amphotericin B. A small amount of culture medium was also added to each insert cup to keep the apical surfaces of the explants hydrated. The explants were incubated for at least 3 h (37°C in 95% O2 and 5% CO2) before ex vivo infection.
Parasite preparation and infection of epithelial cells.
Purified C. parvum oocysts (Iowa isolate) were purchased from the University of Arizona and stored at 4°C in an antibiotic solution (0.01% Tween 20 containing 100 U/mL penicillin and 100 g/mL gentamicin). Before infection of epithelial cells, oocysts were pretreated using a previously described protocol [18] with modifications. Briefly, oocysts were washed with 0.15 mol/L PBS (pH 7.2), centrifuged (3000 g for 15 min at 4°C), and treated with acidic H2O (pH 2.5; 20 min, with vortexing every 5 min, at 37°C). The oocysts were centrifuged (3000 g for 4 min at 20°C), resuspended in parasite-maintenance medium (DMEM containing 4.5 g/L D-glucose, 0.58 g/L L-glutamine, 3.7 g/L sodium bicarbonate, 0.20 g/L bovine bile, 0.004 g/L folic acid, 0.001 g/L 4-aminobenzoic acid, 0.004 g/L D-calcium pantothenate, 0.88 g/L ascorbic acid, 1% heat-inactivated FBS, 2.4 g/L HEPES, 100,000 U/L penicillin, 100,000 g/L streptomycin, and 250 g/L amphotericin B, adjusted to pH 7.4), and incubated for 3 h at 37°C. Intact and empty oocysts were counted to determine the percentage excystation. Epithelial monolayers were washed and replenished with fresh medium. Pretreated oocysts were added to the flasks (1.2 × 105 oocysts/cm2; ratio of oocysts to epithelial cells, 1 : 1; overnight at 37°C in 5% CO2). Ileal explants were infected by adding pretreated oocysts (2.5 × 105 oocysts/cm2 of tissue) to each culture well and incubated overnight (37°C in 95% O2 and 5% CO2).
Cytotoxicity assays.
Cytolytic activity was measured by 51Cr-release assay. The target cells, C. parvuminfected and uninfected HCT-8 and Caco-2 cells, were prepared by disrupting confluent monolayers with a nonenzymatic cell-dissociation solution (Sigma Diagnostics). After washing, the target cells (2 × 106 cells/mL) were labeled with 100 Ci of Na2(51Cr)O4/mL (ICN Biomedicals) in complete medium (2 h at 25°C). The cells were washed 3 times, counted, and resuspended in complete medium. The labeled targets (1 × 104 cells/well) were added to 96-well plates. Cultured effector PBMCs (pretreated with rIL-15 or medium for 72 h) were washed and resuspended in complete medium and added to the wells. The plates were centrifuged and incubated (4 h at 37°C). Released 51Cr was measured in the supernatants by use of a gamma counter (Auto-gamma 5780; Packard Instrument Company). Spontaneous lysis was determined from wells containing target cells in medium only; maximal lysis was determined from target cells lysed with 1% SDS solution. Results from triplicate wells were averaged, and specific cytotoxicity was calculated using the following formula: [(mean cpm experimental - mean cpm spontaneous)/(mean cpm maximum - mean cpm spontaneous)] × 100.
Flow cytometry studies.
Lymphocytes were washed and resuspended in PBS containing 2% heat-inactivated FBS. Cells (5 × 104) were stained using the following monoclonal antibodies (mAbs) at concentrations recommended by the manufacturers (30 min at 4°C): fluorescein isothiocyanate (FITC)labeled antihuman CD3 (Immunotech), phycoerythrin (PE)labeled antihuman CD16 (Pharmingen), PE-labeled antihuman CD56 (Pharmingen), PE-Cy5labeled anti-human CD16 (Pharmingen), PE-Cy5labeled antihuman CD56 (Pharmingen), and PE-labeled antihuman NKG2D (R&D). After staining, the cells were washed, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry (Coulter EPICS XL-MCL; Beckman Coulter).
For intracellular IL-15 staining, HCT-8 cells (1 × 106) were washed with cold PBS and fixed with BD Cytofix/Cytoperm (PharMingen) (20 min at 25°C), washed, and resuspended in BD Perm/Wash (PharMingen). Rabbit antihuman IL-15 (Chemicon) or rabbit IgG (control) was added to the cells (20 min at 4°C). The cells were washed with BD Perm/Wash. After incubation in antirabbit-FITC (20 min at 4°C), cells were washed and resuspended for analysis.
Quantitation of parasite numbers.
HCT-8 cells were infected as described above. After overnight incubation, HCT-8 target cells (5 × 105) and IL-15treated or untreated PBMCs (5 × 106) were incubated overnight at 37°C in 12-well plates. After disruption of the monolayer, the number of epithelial cells was counted. Additional aliquots were fixed and washed using the methods indicated for intracellular IL-15 staining. The cells were then treated with antibody to C. parvum (Sporoglo; Waterborne; 20 min at 4°C), and examined by immunofluorescence microscopy. The percentage of infected HCT-8 cells was determined for 10 fields at 400× magnification.
Immunohistochemistry.
Ileal explants were fixed in 10% formalin, washed, and immersed in 70% ethanol. Serial sections of the formalin-fixed paraffin-embedded tissue blocks were prepared. After deparaffinization and rehydration, sections were heated in citrate buffer for 5 min. Sections were blocked with 2% normal horse serum and 0.5% casein (20 min at 25°C) and incubated with antimajor histocompatibility complex (MHC) class Irelated molecule A (MICA) mAb [19] for 1 h, followed by 30 min with the biotinylated secondary antibody (Vector Laboratories). Sections were washed, treated with ABC Elite (Vector Laboratories) for 30 min, and developed with 3-amino-9-ethylcarbazole (Vector Laboratories) for 1020 min.
Purification of mRNA and cDNA amplification.
Explant specimens were homogenized in 0.5 mL of TRIzol (Invitrogen), and RNA was isolated in accordance with the manufacturer's protocols. cDNA was synthesized from 1 g of total RNA by use of random hexamer oligonucleotides (Roche Diagnostics), using a PTC-100-96V thermal cycler (MJ Research), as described elsewhere [20]. Amplification of the cDNA was accomplished using a sense primer biotinylated on the 5 terminal nucleotide to facilitate later capture using streptavidin. Sequences were synthesized at Sigma-Genosys (The Woodlands, TX) or Integrated DNA Technologies (Coralville, IA) and are listed in table 1. MICA and MICB sequences were designed on the basis of GenBank sequence information, by use of the Primer3 program (available at: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). Denaturation, annealing, and elongation temperatures for polymerase chain reaction (PCR) were 94°C, 54°C, and 72°C, for 30, 30, and 40 s, respectively. Samples were also subjected to 40 amplification cycles, followed by electrophoresis and ethidium bromide staining.
Bioluminescence assay.
Bioluminescent reverse-transcription (RT)PCR was performed using published methods [21, 22]. Briefly, 5 L of biotinylated PCR product generated during linear amplification was denatured, neutralized, and added to wells of Streptavidin-Microtiter plates (Roche Diagnostics) containing 2 ng of digoxigenin-labeled probe in hybridization buffer (62.5 mmol/L Na2HPO4, 94 mmol/L citric acid, 10 mmol/L MgCl2, 0.125% Tween 20, 0.0625% BSA, 15 mmol/L NaN3; pH 6.5). ChemFlash anti-digoxigenin conjugate (5 ng; Chemicon) was added. After 30 min, the conjugate was detected by measuring integrated flash (469 nm) on an ML 3000 luminometer (Dynatech Laboratories). The apparatus precision was determined using 2.5 × 1010 mol of ChemFlash, resulting in a coefficient of variance of 5.0%. Values shown are normalized to cyclophilin (housekeeping gene) as described elsewhere [23] and related as fold change compared with uninfected explant tissue.
Statistical analysis.
The results are presented as means ± SDs. Statistical significance was determined using Student's t test; differences were considered significant at P < .05.
RESULTS
Induction of increased expression of IL-15 by intestinal epithelial cells in response to C. parvum infection.
Within 2040 h after C. parvum infection, HCT-8 cell expression of intracellular IL-15 increased 2-fold (figure 1). Thus, C. parvum infection increases the expression of IL-15 by intestinal epithelial cells. This is, presumably, a direct result of infection.
Enhancement of lysis of C. parvuminfected cells by IL-15.
PBMCs from donors seronegative for C. parvum were cultured with different concentrations of rIL-15 and used as effector cells in 51Cr-release assays using C. parvuminfected HCT-8 target cells (which lack 2 microglobulin and MHC class I expression) [2426]. IL-15 treatment enhanced the cytotoxic activity of PBMCs in a dose- and effector-to-target cell (E : T) ratiodependent manner (fig. 2A). In this experiment, 30.7% ± 12.3% lysis was noted with effector cells stimulated with IL-15 at 10 ng/mL, compared with 8.3% ± 4.5% lysis with untreated cells (P = .04). Similar results were obtained using infected Caco-2 cells, a cell line expressing MHC class I, as targets (data not shown). Thus, IL-15 enhances PBMC-mediated lysis of C. parvuminfected target cells, and lysis is independent of MHC class I molecules.
Elimination of C. parvuminfected cells by IL-15activated cells.
If the modest lysis of cells noted above is biologically significant, then the proportion of parasites eliminated should decrease more than the proportion of cells lysed nonspecifically. To test for the effects of IL-15 activation on parasite numbers, we counted total and infected epithelial cells after incubation alone or with effector PBMCs (with or without IL-15 activation) (table 2). In the absence of PBMCs, the number of HCT-8 cells increased. In the presence of unstimulated effector PBMCs, the number of epithelial cells did not change significantly. Pretreatment of effector cells with IL-15 led to a 28% decrease in the number of epithelial cells (similar to the percentage lysis by 51Cr-release assay). The percentage of infected cells decreased from 25% without effector cells to 8.1% with IL-15activated effector cells, with an 84% reduction in parasite numbers. This suggests that lysis preferentially targets the Cryptosporidium-infected cells.
Enhancement of expression of stimulatory receptor NKG2D on NK cells by IL-15.
We hypothesized that IL-15mediated NK cell activity was mediated by the activation receptor NKG2D. To test this, NK cells, isolated by negative selection, were incubated with or without rIL-15 and analyzed by flow cytometry for surface expression of NKG2D. At baseline, NKG2D was expressed at low levels by 61% ± 17.8% of NK cells. After 68 h of incubation, 49% ± 22% of NK cells cultured in medium alone expressed NKG2D, compared with 68% ± 17% of those incubated with rIL-15 (10 ng/mL). A population of large granular lymphocytes was first noted after culture. The number of these cells doubled with IL-15 treatment (figure 4). Within that fraction, a population of CD16HiNKG2DHi cells was noted that was not present in the population of fresh cells. This correlates with the NK cell population being shown to have enhanced cytolytic activity [27]. IL-15 treatment also prevented the loss of the CD56Hi population, which has previously been shown to be more sensitive to IL-15mediated proliferation [13], to have higher expression of activation markers, and to produce cytokine products [28]. Thus, these studies confirm that rIL-15 enhances expression of NKG2D by NK cells.
Increase in MICA and MICB expression in human ileal explants, resulting from C. parvum infection.
We hypothesized that Cryptosporidium infection up-regulated MICA and MICB, the ligands for NKG2D. Transformed cells chronically express these ligands. Therefore, we studied fresh ileal mucosa for MICA and MICB expression. When compared with uninfected control tissue from the same donor, infected tissue showed a modest but statistically significant increase in both MIC transcripts (table 3). MICA-specific luminescence intensity increased by 1.38 ± 0.37fold in 3 different experiments in infected ileal explants, and the MICB-specific luminescence intensity increased by 1.30 ± 0.14fold. These observations were also confirmed using the HCT-8 cell line (data not shown).
Although statistically significant, these increases were modest. Since only a minority of the epithelial cells were infected, we examined infected and control explants by immunohistochemistry for MICA expression on infected cells. We noted mild staining of the entire epithelium (figure 5). By contrast, intense staining was limited to villous epithelial cells that were infected (parasites projecting into the lumen) and the crypt regions of infected villi. Thus, increased expression of the NKG2D ligands MICA and MICB by ileal epithelial cells was limited to cells infected with C. parvum.
DISCUSSION
In this study, we have evaluated the effect of IL-15 treatment on the ability of PBMCs to lyse C. parvuminfected intestinal epithelial cells. We have demonstratedfor the first time, to our knowledgethat cytolytic cells can lyse C. parvuminfected cells, that IL-15 activates PBMC lysis, and that lysis is mediated by NK cells. IL-15 treatment enhanced the expression of the activation receptor NKG2D on NK cells, particularly on the CD16+ cytolytic subset. Moreover, expression of the NKG2D ligands MICA and MICB was up-regulated in experimentally infected intestinal cell lines and ileal explant tissue, with high levels of expression in infected cells. Taken together, these data show that IL-15 has an important role in clearing Cryptosporidium infection in humans via the activation of NK cells through the NKG2D receptor-ligand system.
We previously reported that experimental human infection with C. parvum leads to increased expression of IL-15 in intestinal tissues [9]. In the present study, we have demonstrated that C. parvum infection augments intracellular IL-15 expression in a human intestinal cell line. Similarly, quantification of IL-15 protein from human jejunal biopsies has revealed that levels of IL-15 are increased within hours of parasite exposure and are maintained through the following week (authors' unpublished data). The levels of IL-15 demonstrated are similar to the levels used in the present study. This pattern of IL-15 expression in human infection differs from that seen in bovine infection [29].
In the intestine, IL-15 stimulates NK, NK T, and T cells [1416, 30, 31, 32]. After IL-15 treatment, however, both intraepithelial T cells [33] and cells expressing NK markers have a greater killing potential [14, 34]. We have shown that depletion of NK cells from the effector cell population markedly reduces the lysis of C. parvuminfected cells. Thus, our data indicate that IL-15, produced within hours of parasite exposure in vivo, may activate NK cell clearance of Cryptosporidium parasites.
Recent studies have defined the mechanisms involved in NK cell activation. NK cells express both inhibitory and stimulatory receptors that control their reactivity [33, 35]. Engagement of inhibitory receptors with MHC class I molecules provides the negative signals necessary for NK cell suppression [35]. A loss of either the inhibitory receptors or MHC class I molecules leads to NK cell activation. Thus, the lack of expression of MHC class I molecules on HCT-8 cells may have nonspecifically augmented their lysis. Inhibitory signals can be overridden by stimulatory signals initiated through activating receptors. NKG2D is a lectin-like molecule that is associated with both NK cell activation and regulation. Here, we have shown that IL-15 treatment leads to increased expression of NKG2D on NK cells. CD16Hi NK cells had enhanced NKG2D expression after IL-15 treatment. This subset of NK cells has been shown to mediate cytotoxicity [27].
Functional ligands of NKG2D in humans include the MHC class Irelated MICA and MICB molecules [36]. MICA and MICB have limited expression in normal gastrointestinal epithelium but are induced by stress and malignant transformation [37, 38]. Intestinal epithelial cells up-regulate expression of MICA in response to some bacterial pathogens, but expression is dependent on the mechanism of cell invasion [39]. Although HCT-8 cells constitutively produce MICA and MICB, C. parvum infection further increased expression in both HCT-8 cells and ileal explant tissue. The relative increase in MICA and MICB expression was modest but highly reproducible and was associated with infected cells. Thus, the increased levels of both NKG2D and MICA and MICB may be critical for the NK celldependent destruction of infected enterocytes. Interestingly, mice do not have homologues for MICA and MICB, which may explain species differences in clearance of C. parvum. For example, parasite clearance in mice appears to be more dependent on IFN- than is parasite clearance in humans.
Despite extensive studies on mechanisms of NK cell activation, relatively little is known about the role of NK cell function in human host defenses against infections. NK cells play an established role in infections, such as those with cytomegalovirus and Chlamydia trachomatis [40, 41]. IL-15 activates and maintains innate and adaptive immune effector cells, induces chemokine secretion by neutrophils and monocytes, and activates these cells to phagocytize and kill bacterial and fungal organisms [42, 43]. In rodent models of intracellular bacterial infections, IL-15 attracts NK cells to infected sites and limits bacterial colonization [4446]. NK cells have also been studied in murine models of malaria and toxoplasmosis [4749].
There are limited data on the role of NK cells in Cryptosporidium infection. Mice deficient in NK cells, such as neonatal SCID mice and mice with the beige mutation, are more susceptible to Cryptosporidium infection than are control mice [5053]. In contrast, animals treated with anti-asialo GM1 antiserum, which eliminates systemic but not intestinal NK cells, do not develop chronic infections [2, 54, 55]. None of these studies examined NK cell function. Interestingly, patients with AIDS, a group at high risk for uncontrolled cryptosporidiosis, have been noted to have defective IL-15 production [56].
In our study, we found that IL-15 stimulated PBMCs to lyse C. parvuminfected cells at concentrations within the physiologic range (other studies have used 1100 ng/mL). Treatment with IL-15 increased expression of NKG2D and enhanced lysis of Cryptosporidium-infected epithelial cells. Furthermore, MICA and MICB expression was significantly increased in infected cells. These data are the first evidence for a role of NK cells in defense against an intestinal parasitic infection in humans, and they suggest that the importance of NK cells in clearing other intracellular pathogens may be underestimated.
Acknowledgments
We thank Sam Gambarin, Katherine Liscum, and Thomas Granchi, for providing intestinal tissue, and David Corry for critical review of the manuscript.
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