TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation

Animals

Three-six month old male ND4 Swiss mice (Harlan Laboratories, Indianapolis, IN) were housed in Macalester College’s research animal facility with a 12-hour light/dark cycle and free access to food and water. A total of 73 mice were used for the experiments shown here (21 controls; 52 experimental) to ensure that appropriate statistical analysis could be performed on data acquired from these experiments. Macalester College’s Institutional Animal Care and Use Committee approved all experimental procedures (Protocol B08Su1).

Drug administration

All drugs were administered using 0.9% saline (VWR, Radnor, PA) vehicle or phosphate buffered saline (EMD Millipore, Billerica, MA). Mice received bilateral intra-plantar (i.pl.) treatments with c48/80 (1.5μg/paw; 10μl; (Sigma-Aldrich, St. Louis, MO)) or saline alone as previously described². Either 200μg/kg of anti-TNF-α neutralizing antibody (R&D Systems, Minneapolis, MN, Polyclonal Goat IgG, Catalog #: AB-410-NA) or 200μl vehicle was injected intravenously 30 minutes prior to c48/80 injection in a protocol adapted from Rocha et al.¹². Rocha et al. administered anti-TNF-α antibodies i.v. 5 mins prior to carrageenan treatment in a mouse model of mechanical hyperalgesia¹².

Thermal sensitivity assessment

To assess thermal sensitivity, single mice treated with i.pl. c48/80 or vehicle were placed in a Plexiglas cylinder on a hotplate analgesia meter (Harvard Laboratories, Edenbridge, KY) maintained at 51.0 ± 0.5°C and removed when prolonged retraction, flipping/licking of the hind paw, or jumping with both hind paws off the hotplate were observed, but no later than 40 seconds, as previously described². Two baseline hotplate latencies were taken 24 and 48 hours before the experiment. Mice with >10 second differences between baselines or <15 second averages were excluded from the experiment. Nociceptive thermal sensitivity was quantified by subtracting the mean baseline thermal latency from the experimental thermal latency at each time point for each mouse.

Paw edema measurements

Change in hind paw width measured using digital calipers (±0.1mm; VWR, Radnor, PA) was calculated as an average of the left and right paw widths. Baseline paw widths for each mouse were taken pre-treatment and subtracted from post-treatment paw widths to calculate tissue edema as previously described².

Myeloperoxidase measurements

Footpads were extracted from hind paws of mice euthanized by CO₂ inhalation, weighed, frozen at -80°C in 5.6μl/mg tissue weight of 50mM K₂HPO₄ buffer (pH 6.0) (Sigma-Aldrich, St. Louis, MO) containing 0.05% hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich, St. Louis, MO), thawed, homogenized in 5x the storage volume of HTAB buffer, sonicated with a 550 Sonic Dismembrator (Fisher Scientific, Waltham, MA), freeze-thawed, and centrifuged (AllegraX-15R; Beckman Coulter, Inc., Pasadena, CA) as previously described^2,13. Absorbance was recorded using a BioTek PowerWave XS plate reader (BioTek, Winooski, VT) at 450nm after a 20-minute incubation in 50mM phosphate buffer (pH 6.0) with 0.025% hydrogen peroxide (Sigma Aldrich, St. Louis, MO) and 0.167mg/ml o-dianisidine-dihydrochloride (Sigma Aldrich, St. Louis, MO) at room temperature in the dark. Myeloperoxidase (MPO) levels were normalized to tissue weight and presented as OD/g of wet tissue.

TNF-α measurements

For protein and gene expression studies, hind paws were excised from mice euthanized by CO₂ inhalation, flash-frozen in liquid nitrogen and stored at -80°C.

For protein studies, flash-frozen paws were homogenized in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) supplemented with protease inhibitor (Cocktail Set IV; EMD Biosciences, Billerica, MA) using a Tissue-Tearor (BioSpec; Model 985370). Homogenates were incubated on ice for 20 minutes, centrifuged at 2000 rpm for 10 minutes at 4°C, and lysate supernatants stored at -80°C. We quantified TNF-α cytokine levels by ELISA according to the manufacturer’s instructions (eBioscience, San Diego, CA).

To measure cytokine gene expression, total RNA was extracted from flash-frozen plantar tissue (Total RNA Mini Kit, Midwest Scientific, St. Louis, MO), quantified with Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE), and reverse-transcribed in a 2720 Thermal Cycler (Life Technologies, Grand Island, NY) using the Superscript III First-Strand Synthesis System (Life Technologies, Grand Island, NY) with 100ng of RNA per reaction. Relative transcript abundance was determined by quantitative comparative reverse transcriptase PCR (qRT-PCR)¹⁴ using TaqMan Gene Expression Assay Primer/Probe Sets and TaqMan MasterMix (Life Technologies, Grand Island, NY) in a Bio-Rad iCycler (Bio-Rad, Hercules, CA) using β-2-microglobulin (β2m; Mm00437762_m1, Life Technologies, Grand Island, NY) and TNF-α (Mm00443260_g1, Life Technologies, Grand Island, NY) primer sets. Fold expression was normalized to β-2-microglobulin levels and calculated as previously described^14,15.

Histology

Excised hind paws (from mice euthanized by CO₂ inhalation) were fixed in 10% buffered formalin (VWR, Radnor, PA) for 24 hours, transferred to 70% ethanol (Sigma-Aldrich, St. Louis, MO), decalcified for 1–2 weeks in 15% EDTA, hydrated, and embedded in paraffin. 4μm sagittal sections were stained with toluidine blue (Tb) at pH 2.3 for 3 minutes (Sigma-Aldrich, St. Louis, MO) or hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO) for the detection of mast cells or neutrophils, respectively, at 400x magnification with ≥3 biological replicates per treatment. Tb-stained mast cells were counted in 10 fields/section and degranulation was scored based on the number of granules observed outside the boundary of the cell: intact (0), mild (1–10), moderate (10–20), and extensive (20+) as previously described². The same investigator performed all counts and was blinded to treatment groups. Neutrophils were imaged between the heel and the toes on the plantar side of the hind paw section using an Olympus CX21LED light microscope and camera (Olympus Corporation of the Americas, Center Valley, PA).

Statistical analysis

Data were processed using Microsoft Excel (Redmond, WA) and graphed with PRISM 5.0 (GraphPad, San Diego, CA). All statistical analyses were performed using JMP 9.0 (SAS, Cary, NC). All data are presented as the mean ±SEM. Data were analyzed using one-way ANOVA, and the Tukey-Kramer HSD post hoc test, at each time point. Statistical significance was defined as p<0.05. Thermal sensitivity and hind paw edema measurements included 15–18 mice per treatment group. Other analyses used 3–9 mice per treatment group. All data represent 2–3 independent experiments.

TNF-α neutralizing antibody blocks thermal hyperalgesia and early tissue swelling induced by compound 48/80-provoked mast cell degranulation in the hind paw tissue of ND4 mice

Male ND4 Swiss mice bilaterally injected with 1500ng of c48/80 in the hind paws showed hyperalgesic withdrawal responses ~10 seconds sooner than their baseline withdrawal 30 minutes after treatment; these responses were resolved by 2.5 hours (Figure 1A). Pre-treatment with an intravenous injection of 200μg/kg TNF-α neutralizing antibody 30 minutes prior to c48/80 injection significantly abrogated these responses at 30 minutes and 1.5 hours after intra-plantar treatment with c48/80; pre-treated mice showed hyperalgesic withdrawal responses <5 seconds sooner than their baseline responses 30 minutes after treatment (Figure 1A). Withdrawal responses of control and anti-TNF-α treated groups were significantly different at 1.5 hours after treatment and indistinguishable at 2.5 hours when the c48/80-provoked hyperalgesic responses were resolved. Anti-TNF-α pretreatment also significantly reduced hind paw edema compared to mice without pre-treatment at 30 minutes but not at 1.5 and 2.5 hours (Figure 1B). Saline-treated control mice had no increase in thermal sensitivity and showed no tissue swelling (Figure 1A–B). Overall, thermal sensitivity was reduced in a more sustained manner compared to tissue edema.

Figure 1. Anti-TNF-α neutralizing antibody abrogates c48/80-induced thermal hyperalgesia.

Mice were pre-treated with 200μg/kg anti-TNF-α neutralizing antibody or vehicle (200μl, i.v.) 30 minutes before bilateral intra-plantar c48/80 or 0.9% saline injections (1.5μg/paw; 10μl). The bars represent the mean change in thermal paw withdrawal latency (A) and the change in paw width (B) from baseline and error bars represent ± SEM. Anti-TNF-α administration abrogated thermal hyperalgesia at 0.5 and 1.5h after c48/80 injection (A) and reduced paw edema significantly at 0.5h (B). Significances are compared to Sal/Sal (# = p<0.05; ## = p<0.001) and Sal/c48/80 (* = p<0.05; *** = p<0.001). n = 12 in Sal/Sal; n = 18 for Sal/c48/80; n = 19 for anti-TNF/c48/80 treatment groups; data are pooled from 2 independent experiments.

To confirm that the antibody pre-treatment did not have an effect on the extent of mast cell degranulation caused by c48/80 that would have consequently affected mast cell-mediated hyperalgesia, we evaluated the levels of degranulation in toluidine blue-stained 4μm paraffin sections as previously described² and confirmed that the levels of mild, moderate and extensive degranulation were not altered in anti-TNF-α treated mice (Figure 2).

Figure 2. Pre-treatment with anti-TNF-α does not affect c48/80-provoked mast cell degranulation.

Mice were pre-treated with 200μg/kg anti-TNF-α neutralizing antibody or vehicle (200μl, i.v.) 30 minutes before bilateral intra-plantar c48/80 injection (1.5μg/paw; 10μl). Mice were euthanized 2.5h after c48/80 administration; their hind paws were harvested, preserved in 10% buffered formalin and ethanol, and 4μm paraffin sections stained with toluidine blue for mast cell visualization. Mast cells were counted at 400x and their degranulation status was assessed as described previously². Bars represent mean percentages of mast cells examined that are assigned to intact or mild, moderate and extensive degranulation status respectively and the error bars represent ±SEM. n = 3 mice per treatment group; data are representative of 2 experiments.

Taken together, pre-treatment with anti-TNF-α neutralizing antibody did not change the extent of local mast cell degranulation in the mouse hind paw tissue but markedly reduced resulting thermal sensitivity and tissue swelling provoked by injection of the basic mast cell secretagogue c48/80.

Intra-plantar c48/80 administration does not induce changes in TNF-α protein or mRNA levels in the hind paw tissue of ND4 male mice

Mast cells contain pre-formed TNF-α in their granules¹⁶ that is released early upon degranulation¹⁶. We analyzed TNF-α protein and mRNA levels in c48/80-treated and untreated hind paw tissue to determine whether there was new synthesis of TNF-α immediately following treatment with the basic mast cell secretagogue. We found that neither protein nor mRNA levels of the cytokine detectably increased in the tissue within 3 hours following intra-plantar c48/80 injection (Figure 3A–B). Thus, it is most likely that the anti-TNF-α neutralizing antibody treatment targets pre-formed, rather than newly synthesized, TNF-α molecules and this blockade contributes to the resulting decrease in thermal sensitivity in the hind paw tissue.

Figure 3. Intra-plantar c48/80 administration does not induce changes in TNF-α protein or mRNA levels in the hind paw tissue of ND4 male mice.

Mice were pretreated with 200μg/kg anti-TNF-α neutralizing antibody or vehicle (200μg, i.v.) 30 minutes before bilateral intra-plantar c48/80 injection (1.5μg/paw; 10μl). 2.5h after c48/80 treatment, their hind paws were excised and preserved for TNF-α protein quantification by ELISA (A). Another set of mice was euthanized at 3h after c48/80 treatment; their footpads were excised for TNF-α mRNA abundance quantification by qRT-PCR (B). Bars represent average pg/ml TNF-α protein (A) and fold-expression of TNF-α transcripts compared to β2m (B) and the error bars represent ±SEM. n = 6 (Sal/Sal); 9 (Sal/c48/80) for ELISA assays and n = 3 mice per treatment group for qRT-PCR; data are representative of 3 (ELISA) and 2 (qPCR) experiments.

TNF-α neutralizing antibody pre-treatment does not affect early neutrophil recruitment in the tissue

Neutrophil influx into the affected tissue contributes to c48/80-provoked hind paw thermal sensitivity² and TNF-α is a known neutrophil attractant^3,17. We have previously shown that the blockade of neutrophil influx can abrogate mast cell-dependent thermal sensitivity in the mouse hind paw². Therefore, we investigated whether pre-treatment with the TNF-α neutralizing antibody had an effect on neutrophil influx. We found that levels of myeloperoxidase (an enzyme indicating the presence of activated neutrophils) in the hind paw tissue of mice injected bilaterally with 1500ng of c48/80 were not significantly reduced with anti-TNF-α pre-treatment (Figure 4A). We further confirmed this by examining the presence of neutrophils in 4μm paraffin embedded hind paw tissue sections stained with H&E and found that infiltrating neutrophils were present in the hind paws of mice that received TNF-α neutralizing antibody (Figure 4B). Neutrophil numbers in the hind paw tissue of mice following c48/80 injection with and without anti-TNF-α pre-treatment were approximately a total of ~150 neutrophils in 10 randomly chosen visualized sections per paw (see Data File). Therefore, pre-treatment with anti-TNF-α neutralizing antibody had little to no inhibitory effect on the levels of tissue infiltrating neutrophils and myeloperoxidase enzyme activity in the hind paw tissue of c48/80-treated mice.

Figure 4. Pre-treatment with anti-TNF-α does not prevent c48/80-provoked neutrophil influx.

Mice that received c48/80 intra-plantar injections following anti-TNF-α pretreatment did not have significantly different levels of myeloperoxidase enzyme activity compared to mice that received vehicle pretreatment (A). Hind paws were harvested from euthanized mice and assayed for MPO activity; bars represent average MPO activity as OD/g wet tissue and error bars represent ±SEM (A). Mice were euthanized 2.5h after c48/80 administration, their hind paws were harvested, preserved in 10% buffered formalin and ethanol, and 4μm paraffin sections stained with hematoxylin & eosin for neutrophil visualization at 1000x. Similar to c48/80-treated mice (B), mice pre-treated with anti-TNF-α neutralizing antibody showed clear evidence of neutrophil influx (indicated by black arrowheads) into the affected hind paw tissue (C). n = 3 mice per treatment group for MPO assay; data are representative of 2 experiments.