The promonocytic human cell line U-937 (American Type Culture Collection, ATCC, Manassas, VA, USA) was propagated in RPMI-1640 endotoxin-free medium (In vitro, Life Technologies, Eggenstein, Germany) supplemented with 2mM l-glutamine, 1.5g/L sodium bicarbonate, 4.5g/L d-glucose, 10mM HEPES, 1.0mM sodium pyruvate and 10% heat-inactivated, endotoxin-free fetal bovine serum (FBS, Gibco BRL Life Technologies, Grand Island, NY, USA). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. PBMC were obtained from healthy individuals or patients with AR by Böyum method.13 Briefly, 4mL of diluted blood sample was carefully layered on 3mL of Ficoll-Paque PLUS (GE Heathcare Bio-Sciences Corp, Piscatawa, NJ, USA), tubes were centrifuged at 400×g for 30min at room temperature. Using a Pasteur pipette we take off the mononuclear layer and washed it with balanced salt solution. Typically recovered cells were ≥95% mononuclear cells with ≥95% viability.

Time-course experiments were carried out in triplicate groups of 4×106mL−1. U-937 cells incubated at 37°C in a humidified atmosphere with 5% CO2 during 0–10h were treated as follows: (a) in RPMI-1640 medium alone (unstimulated); (b) MLIF (50g/mL) added (per se effect); (c) LPS (10 ng/mL) added (induced expression), and (d) MLIF and LPS added simultaneously (interference). After 0.3, 1, 2, 4, 6, 8 and 10h, cells were harvested by centrifugation. All cell pellets were used for RNA isolation, and 4, 6 and 8h supernatant fluids employed for IL-1β immunoreactivity. Triplicate groups of 4×106mL−1 human PBMC were incubated during 4 and 6h without stimulus, MLIF, LPS or both. After 4h, the cell pellet (for RNA isolation) or after 6h, the supernatant fluid (for IL-1β immunoreactivity) was harvested by centrifugation. At the conclusion of all experiments, cell viability was ≥90% by trypan blue dye (Sigma Chemical Co., St. Louis, MO) exclusion. The concentration of MLIF employed in these studies was based on previous in vitro functional experiments.2,3 Simultaneous addition of MLIF to assays was employed, because pilot studies have revealed that MLIF mainly affected cytokine induction when added prior to (weak) or simultaneously (strong), but less after LPS stimulation.

A single batch, 96% pure MLIF (Met-Gln-Cys-Asp-Ser, MQCNS) endotoxin-free sample was commercially obtained (American Peptide Co., Sunnyvale, CA, USA). MLIF was dissolved in sterile PBS and tested for endotoxin activity using Limulus amoebocyte lysate KTA (Charles River Endosafe Inc., Charleston, SC, USA). Endotoxin activity was ≤0.0625UIEmL/endotoxin. LPS Escherichia coli serotype 0111:B4 was obtained from Sigma.

Females, 35–59 years of age, were recruited from the Blood Bank (healthy donors) and Rheumatology Clinic of “Dr. Emilio Varela-Lujan” General Hospital, Mexican Institute of Social Security (IMSS) in Zacatecas City, Mexico. Patients with RA were classified as such when they fulfilled four or more American College of Rheumatology (ACR) criteria. Subjects were under Anti-inflammatory non-steroid treatment (AINES), or glucocorticoids at doses ≤10mg/day and were free of biologic therapy (anti-TNFα, anti-IL-1β or anti CD20) at least 6 months prior to the study. The study included 13 healthy subjects and 11 patients with RA. All study participants gave written informed consent to participate in the study. The procedure complies with the international ethics for human research and was approved by the Dr. Emilio Varela Luján Hospital Ethics Committee.

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) as described.3 RNA purity was established in 2.5% denaturing agarose gels, and the concentration determined in a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA). For reverse transcription, 1μg of total RNA was mixed with 1μL oligo-dT (12–18-mer, Invitrogen), 200U SuperScript™ reverse transcriptase (Invitrogen), 1× First Stand Buffer (Invitrogen), and 0.5mM mixed dNTPs (dATP, dCTP, dGTP, dTTP) (Promega, Co., Madison, WI, USA). The mixture was incubated at 42°C for 1h. Reverse transcription was heat-inactivated at 80°C for 10min and the resulting cDNA was employed for real-time polymerase chain reaction (PCR).

Real-time PCR was performed in capillaries in a 20μL reaction mix containing Light Cycler Taqman (Roche Applied Science, Salt Lake City, UT, USA), 0.2μM specific primer mix, and 0.1μM of gene-specific hydrolysis probes from the Universal Probe Library (Roche) and cDNA. PCR reaction included 45 cycles consisting of denaturation at 95°C for 10s, annealing at 60°C for 20s, and extension at 72°C for 1s. All reactions were performed in a LightCycler 2.0 instrument (Roche) and data analysis was conducted with LightCycler software 4.05 (Roche).

We obtained the relative expression of IL-1β induced by LPS, MLIF and LPS+MLIF with respect to unstimulated cells using the ΔΔCP method.14 Briefly, we copied the CP values for each sample in an Excel spreadsheet, the first delta CP was calculated from IL-1β CP minus glyceraldehyde 3-phosphate dehydrogenase (GAPDH) CP for each sample accordingly. Deltadelta CP for each condition was equal to stimulated sample delta CP minus unstimulated sample CP. Relative expression was calculated using Relative expression=2(−deltadelta CP) formula.

We considered IL-1β as target gene and GAPDH as housekeeping gene.

For comparison among study groups (healthy subjects vs patients with RA), real-time PCR calibration curves for IL-1β and GAPDH genes were produced from purified specific amplicons. In all groups we obtained normalized expression ratio (ner) of each condition (unstimulated, LPS, MLIF and LPS+MLIF) employing the formula:

Comparison among different ner was used to evaluate basal IL-1β, and responses induced by LPS and MLIF, as well as MLIF capacity to antagonize LPS response.

IL-1β proteins in supernatant fluids were measured using a commercial ELISA kit DuoSet® (R & D Systems, Inc., Minneapolis, MN, USA). Plates were incubated overnight at 4°C, washed and blocked. Final development employed ExtrAvidin™ HRP and substrate 3,3′,5,5′-tetramethyl-benzidine (Sigma). Microtiter plates were read at 450nm in a BIO-RAD microplate Benchmark densitometer (Richmond, CA, USA). Results were expressed as ng/mL. Sensitivity for IL-1β was ≥2pg/mL.

Results are expressed as mean ranks. Statistical significance of differences in mean values between different groups was evaluated by Kruskal–Wallis test, followed by U-Mann–Whitney test for inter-group comparisons. A value of p<0.05 was considered to be significant. All analyses were performed with SPSS software version 15.0 (SPSS Inc., Chicago, IL).

Previous work utilizing U-937 demonstrated that MLIF was able to decrease PMA-induced expression of chemokines MIP-1α and MIP-1β, as well as that of cytokine IL-1β.2,3 We observed this effect only when the peptide was administered prior to or simultaneously with the activator agent. The neutralizing effect of MLIF on such an event was outstanding, if we take into account that PMA is a powerful cell activator that disrupts several cell signaling pathways. On the other hand, because MLIF is involved in the NF-κB signaling, we were interested in observing the amebic peptide effect on the production of LPS/TLR4 signaling-dependent IL-1β.

In a triplicate time-course experiment using U-937 cells, LPS-induced expression of IL-1β was followed with real-time PCR. Specific IL-1β mRNA was detected in a 1–10h window of time (Fig. 1). Maximum response to LPS peaked at 4h post-stimulation with 94±1.15 (mean±SEM) folds, and afterwards there was a symmetrically decreasing production. When MLIF was administrated simultaneously with LPS, the amebic peptide exhibited an astonishing antagonist effect at 4h (relative expression 94±1.15-fold with LPS vs 8±0.03-fold with LPS+MLIF (p<0.05)). On the other hand, amebic peptide per se showed no IL-1β regulatory capacity.

Figure 1.

Time-course expression of IL-1β in U-937 cells. Triplicate cultures of 4×106 U-937 cells in the presence of MLIF, LPS or both were harvested at 0.3, 1, 2, 4, 6, 8 and 10h. RNA was obtained from pellets and used for real time PCR. Comparative expression was established using unstimulated cells as reference. LPS induced a strong up-regulation of IL-1β which peaks at 4h. MLIF per se did not affect IL-1β basal expression but MLIF was able to reduce significantly the stimulatory effect of LPS when amebic peptide was added simultaneously to LPS (p=0.05). Bars of SEM are <1 in all points.

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Supernatants from mentioned triplicate experiment were used for measuring IL-1β immunoreactivity released by U-937 cells stimulated with LPS, MLIF or both at three different times (4, 6, and 8h). A higher amount of IL-1β was detected after 6h of incubation where LPS-induction was maximum with 18.5±0.74pg/mL, statistical difference between LPS vs MLIF (18.5±0.4 vs 13.2±0.4), and LPS vs unstimulated cells (18.5±0.4 vs 9.6±0.5) was significant with p<0.05 for both. MLIF showed no effect on IL-1β release by LPS stimulation. At 4 and 8h IL-1β response to LPS was poor, and in most conditions immunoreactivity was near the assay detection limit (Fig. 2). There is a trend for MLIF to interfere with LPS effect at 4h and 8h but statistics showed it not to be significant. Once again, the peptide per se did not affect the production of IL-1β.

Figure 2.

Time-course of IL-1β immunoreactivity in U-937 cells. Triplicate cultures of 4×106 U-937 cells in the presence of MLIF, LPS or both were harvested at 4, 6 and 8h by centrifugation. Supernatant fluids were employed for IL-1β immunoreactivity. In general IL-1β released to medium was low at 4 and 8h, response to LPS was higher at 6h *(p=0.05), MLIF per se had no effect over IL-1β release and did not interfere with LPS stimulation.

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We were interested in testing the observed MLIF effects in differentiated immunocompetent cells. PBMC cultures from healthy subjects and patients with RA were used to look at basal expression of IL-1β. As expected, patients with RA exhibited higher expression of IL-1β mRNA in comparison with healthy subjects. 91% of patients with RA (10 of 11 patients) IL-1β ner was >1; meanwhile in 54% of healthy subjects (9 of 13) IL-1β ner was >1 (Fig. 3a). Statistical data showed a significantly higher expression of IL-1β in patients with RA compared with healthy donors (p=0.03).

Figure 3.

Effect of MLIF in PBMC from healthy and patients with RA. Healthy donors (n=13) and patients with RA (n=11) PBMC were cultured during 4h in the presence of LPS, MLIF or both. qPCR analysis for IL-1β transcripts was carried out in each case and comparison between groups established by a normalized expression ratio (ner). (a) Patients with AR constitutively express more IL-1β than healthy donors. IL-1β ner between them was statistically different (p=0.03). (b) MLIF shows a down-regulatory effect on IL-1β expression in patients with RA which ner is <1 in 91% of cases (p=0.01). (c) Capacity of LPS to up-regulate IL-1β in both healthy donors and patients with RA was not significantly different. (d) MLIF was able to down-regulate LPS-induction in all patients with RA (p=0.001).

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The capacity of MLIF to affect the expression of IL-1β was determined as the ratio of MLIF ner to unstimulated ner. The presence of MLIF in PBMC cultures decreased IL-1β expression (ratio <1) in 91% of patients with AR (10 of 11) and in 46% of healthy donors (6 of 13) (p=0.01) (Fig. 3b).

Ratios of LPS-induced ner to unstimulated ner were employed to evaluate LPS stimulatory capacity in PBMC cultures. We considered an increase of >2-folds in IL-1β expression as a biologically effective criterion. Studied groups responded in similar fashion to LPS stimulus; 64% (7 of 11) patients with RA and 62% (8 of 13) healthy donors responded to LPS, and no significant differences were found among them (Fig. 3c).

We considered that MLIF could antagonize with LPS when ratios of LPS+MLIF ner to LPS-induced ner were <1. Expression of IL-1β decreased in 100% (11 of 11) of patients with RA and in 38% (5 of 13) of healthy subjects (p=0.001) (Fig. 3d).

IL-1β inmunoreactivity in PBMC cultures was in general low (below the detection limit in the majority of cases). It was only possible to detect IL-1β in 36% (4 of 11) of patients with RA and in 8% (1 of 13) of healthy donors. Apparently, RA group releases more IL-1β than healthy group (18±2.1 vs 8±1.6pg/mL, respectively). However, because the low number of data, no significant differences were found between them. In the group of patients with RA, we observed a MLIF trend to diminish the amount of IL-1β spontaneously released in culture from 18.0 to 5.1pg/mL, as well as an antagonist effect on LPS-stimulation in both RA and healthy groups (from 14.6 to 9.0; and from 19.2 to 7.1pg/mL, respectively) (Fig. 4). This result is interesting but not conclusive due the low number of immunoreactive samples.

Figure 4.

Immunoreactivity of IL-1β in PBMC from healthy donors and patients with RA IL-1β immunoreactivity was detected by ELISA in supernatant fluids of 6h cultures of PBMC from healthy donors (n=11) and patients with RA (n=13). IL-1β was not detected in most patients with RA and healthy donors. Patients with RA show higher spontaneous release of IL-1β than healthy donors, and a poor response to LPS stimulus. MLIF per se seems to down-regulate IL-1β in patients with RA, and interferes with LPS induction in both healthy donors and patients with RA. Statistics were not available because of the low number of immunoreactive samples.

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