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Novel method of monitoring trace cytokines and activated STAT molecules in the paws of arthritic mice using multiplex bead technology.

Lu LD, Stump KL, Seavey MM - BMC Immunol. (2010)

Bottom Line: How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained.Local cytokine responses could be matched with serum cytokine levels and joint pathology results.STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Worldwide Discovery Research, Cephalon, Inc, West Chester, Pennsylvania 19380, USA.

ABSTRACT

Background: The use of mouse models to study human disease provides useful data that can provide support for research projects or an existing drug discovery program. How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained. Designing new treatments for rheumatic diseases, such as rheumatoid arthritis (RA), requires complex immunocompetent models that depend on intricate cytokine networks. Using local cytokines, signal transduction and transcription factor molecules as potential biomarkers to monitor disease and treatment efficacy is the best method to follow the progression of tissue damage and repair when testing an unknown compound or biologic. Described here in this report, a novel method for the non-enzymatic extraction and measurement of cytokines and signal transducers and activators of transcription (STAT) molecules using Luminex® bead array technology in two different mouse models for human RA--collagen antibody-dependent arthritis (CAIA) and collagen-induced arthritis (CIA).

Results: Dynamic expression of several pro-inflammatory cytokines responsible for promoting disease augmentation overtime were monitored, such as IL-1β, TNFα, IL-6 and IL-12, locally in the paws of affected animals directly ex vivo. Local cytokine responses could be matched with serum cytokine levels and joint pathology results. In addition, STAT1, 3, and 5a/b activation status could be monitored with confidence using specifically formulated extraction buffer that protected the phosphorylation site. STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone. Here reported a novel method of extracting joint fluid from the paws of inflamed mice coupled with powerful multiplex bead technology allowing us to measure cytokine responses, pharmacodynamic markers such as STATs and pharmacokinetic analysis of dosed agent all from the same sample directly ex vivo.

Conclusions: This method is powerful in that it is applicable to multiple autoimmunity model types, streamlines ex vivo readouts in a high-throughput manner, and allows multiplexing providing the investigator with an array of options and possible analytes when developing preclinical animal models to support drug discovery efforts in the search for new treatments for rheumatic diseases.

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Paw Cytokines Follow Disease Progression in CAIA Mouse Model. Female DBA/1 mice were injected i.v. with anti-collagen type II antibodies and given a LPS booster injection three days after the antibody transfer. This combination of antibodies and adjuvant provides a potent and rapid, but self-limiting, antibody dependent arthritis. (A) Disease is established and mice enter the study when specific criteria are met (see materials and methods). Dexamethasone significantly reduced mean paw thickness (averaged from individually measured paws - front, back, right and left), *p < 0.01 versus vehicle control group, N = 10 per group. (B) Paw IL-1β concentration sampled in 3 mg of total protein prepared as described in the materials and methods. As expected from the paw swelling, dexamethasone reduces cytokine levels compared to the vehicle control group, *p < 0.05. (C) Paw IL-12 slowly increases overtime as disease progresses, dexamethasone treatment significantly reduces paw cytokine levels, *p < 0.05, N = 5 per group. (D) Histological analysis of paw swelling using H&E and Safranin-O stains (carpus shown). Vehicle shows leukocytic infiltrates, matrix degradation and osteolysis, dexamethasone treated mice compared to non-diseased animals (data not shown), N = 5 mice per group, representative images shown, 4× magnification. All graphs show Mean ± SEM.
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Figure 2: Paw Cytokines Follow Disease Progression in CAIA Mouse Model. Female DBA/1 mice were injected i.v. with anti-collagen type II antibodies and given a LPS booster injection three days after the antibody transfer. This combination of antibodies and adjuvant provides a potent and rapid, but self-limiting, antibody dependent arthritis. (A) Disease is established and mice enter the study when specific criteria are met (see materials and methods). Dexamethasone significantly reduced mean paw thickness (averaged from individually measured paws - front, back, right and left), *p < 0.01 versus vehicle control group, N = 10 per group. (B) Paw IL-1β concentration sampled in 3 mg of total protein prepared as described in the materials and methods. As expected from the paw swelling, dexamethasone reduces cytokine levels compared to the vehicle control group, *p < 0.05. (C) Paw IL-12 slowly increases overtime as disease progresses, dexamethasone treatment significantly reduces paw cytokine levels, *p < 0.05, N = 5 per group. (D) Histological analysis of paw swelling using H&E and Safranin-O stains (carpus shown). Vehicle shows leukocytic infiltrates, matrix degradation and osteolysis, dexamethasone treated mice compared to non-diseased animals (data not shown), N = 5 mice per group, representative images shown, 4× magnification. All graphs show Mean ± SEM.

Mentions: Since serum cytokines are difficult to measure using this model [12,13] and since the most reflective disease activity can be found locally in the paw [18] building an extraction assay whereby cytokines are collected from inflamed paws and read on a multiplex bead assay using the powerful Luminex® technology would be greatly beneficial. As illustrated in the method schematic flow chart (Figure 1), front and back paws are collected and cut into small pieces (2 mm × 2 mm) then snap frozen and stored at -80°C until ready for processing. Keeping everything on dry-ice, samples were homogenized and cellular/joint extracts were analyzed for either cytokine or phosphorylated STAT activation. Using Dexamethasone at 1.5 mg/kg body weight we were able to validate our method modulating the local cytokine milieu using a standard-of-care option, (i.e., glucocorticoid treatment). Established arthritis was generated and glucocorticoid, dexamethasone, was given three times a week, i.p., and reduced paw swelling measured via a digital caliper (Figure 2A, *p < 0.01) accompanied by a clinical score (Additional file 1, Figure S1A). Body mass did not change between treatment and vehicle group (Additional file 1, Figure S1B). Early model optimization data was collected but is not shown here for clarity. Serum cytokine levels were inconsistent and in many cases undetectable as expected (data not shown). However, using this extraction method both early (Figure 2B, IL-1β) and late (Figure 2C, IL-12) cytokines could be detected. Cytokine IL-1β, one of the signature cytokines of RA and a target of a commonly used RA therapeutic [19,20], is found shortly after arthritis induction and can be reduced upon dexamethasone treatment (Figure 2B, *p < 0.05). Similar findings were found for IL-6 and IL-1β using conventional RT-PCR methods (data not shown). The source for IL-1β are usually activated macrophage, dendritic cells and NK cells [21]. The source for IL-12 are usually tissue activated macrophage (F4/80+) and dendritic cells but can also be made by B-cells [22,23]. IL-12 is usually observed following early innate cytokines including TNFα and IL-6 [24,25]. Cytokines TNFα and IL-2 were also reduced upon dexamethasone treatment (Additional file 1, Figure S1C-D). This reduction in inflammatory cytokines corresponded with reduced inflammation and cartilage degradation as demonstrated in paw histological H&E and Safranin-O sections (Figure 2D).


Novel method of monitoring trace cytokines and activated STAT molecules in the paws of arthritic mice using multiplex bead technology.

Lu LD, Stump KL, Seavey MM - BMC Immunol. (2010)

Paw Cytokines Follow Disease Progression in CAIA Mouse Model. Female DBA/1 mice were injected i.v. with anti-collagen type II antibodies and given a LPS booster injection three days after the antibody transfer. This combination of antibodies and adjuvant provides a potent and rapid, but self-limiting, antibody dependent arthritis. (A) Disease is established and mice enter the study when specific criteria are met (see materials and methods). Dexamethasone significantly reduced mean paw thickness (averaged from individually measured paws - front, back, right and left), *p < 0.01 versus vehicle control group, N = 10 per group. (B) Paw IL-1β concentration sampled in 3 mg of total protein prepared as described in the materials and methods. As expected from the paw swelling, dexamethasone reduces cytokine levels compared to the vehicle control group, *p < 0.05. (C) Paw IL-12 slowly increases overtime as disease progresses, dexamethasone treatment significantly reduces paw cytokine levels, *p < 0.05, N = 5 per group. (D) Histological analysis of paw swelling using H&E and Safranin-O stains (carpus shown). Vehicle shows leukocytic infiltrates, matrix degradation and osteolysis, dexamethasone treated mice compared to non-diseased animals (data not shown), N = 5 mice per group, representative images shown, 4× magnification. All graphs show Mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Paw Cytokines Follow Disease Progression in CAIA Mouse Model. Female DBA/1 mice were injected i.v. with anti-collagen type II antibodies and given a LPS booster injection three days after the antibody transfer. This combination of antibodies and adjuvant provides a potent and rapid, but self-limiting, antibody dependent arthritis. (A) Disease is established and mice enter the study when specific criteria are met (see materials and methods). Dexamethasone significantly reduced mean paw thickness (averaged from individually measured paws - front, back, right and left), *p < 0.01 versus vehicle control group, N = 10 per group. (B) Paw IL-1β concentration sampled in 3 mg of total protein prepared as described in the materials and methods. As expected from the paw swelling, dexamethasone reduces cytokine levels compared to the vehicle control group, *p < 0.05. (C) Paw IL-12 slowly increases overtime as disease progresses, dexamethasone treatment significantly reduces paw cytokine levels, *p < 0.05, N = 5 per group. (D) Histological analysis of paw swelling using H&E and Safranin-O stains (carpus shown). Vehicle shows leukocytic infiltrates, matrix degradation and osteolysis, dexamethasone treated mice compared to non-diseased animals (data not shown), N = 5 mice per group, representative images shown, 4× magnification. All graphs show Mean ± SEM.
Mentions: Since serum cytokines are difficult to measure using this model [12,13] and since the most reflective disease activity can be found locally in the paw [18] building an extraction assay whereby cytokines are collected from inflamed paws and read on a multiplex bead assay using the powerful Luminex® technology would be greatly beneficial. As illustrated in the method schematic flow chart (Figure 1), front and back paws are collected and cut into small pieces (2 mm × 2 mm) then snap frozen and stored at -80°C until ready for processing. Keeping everything on dry-ice, samples were homogenized and cellular/joint extracts were analyzed for either cytokine or phosphorylated STAT activation. Using Dexamethasone at 1.5 mg/kg body weight we were able to validate our method modulating the local cytokine milieu using a standard-of-care option, (i.e., glucocorticoid treatment). Established arthritis was generated and glucocorticoid, dexamethasone, was given three times a week, i.p., and reduced paw swelling measured via a digital caliper (Figure 2A, *p < 0.01) accompanied by a clinical score (Additional file 1, Figure S1A). Body mass did not change between treatment and vehicle group (Additional file 1, Figure S1B). Early model optimization data was collected but is not shown here for clarity. Serum cytokine levels were inconsistent and in many cases undetectable as expected (data not shown). However, using this extraction method both early (Figure 2B, IL-1β) and late (Figure 2C, IL-12) cytokines could be detected. Cytokine IL-1β, one of the signature cytokines of RA and a target of a commonly used RA therapeutic [19,20], is found shortly after arthritis induction and can be reduced upon dexamethasone treatment (Figure 2B, *p < 0.05). Similar findings were found for IL-6 and IL-1β using conventional RT-PCR methods (data not shown). The source for IL-1β are usually activated macrophage, dendritic cells and NK cells [21]. The source for IL-12 are usually tissue activated macrophage (F4/80+) and dendritic cells but can also be made by B-cells [22,23]. IL-12 is usually observed following early innate cytokines including TNFα and IL-6 [24,25]. Cytokines TNFα and IL-2 were also reduced upon dexamethasone treatment (Additional file 1, Figure S1C-D). This reduction in inflammatory cytokines corresponded with reduced inflammation and cartilage degradation as demonstrated in paw histological H&E and Safranin-O sections (Figure 2D).

Bottom Line: How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained.Local cytokine responses could be matched with serum cytokine levels and joint pathology results.STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Worldwide Discovery Research, Cephalon, Inc, West Chester, Pennsylvania 19380, USA.

ABSTRACT

Background: The use of mouse models to study human disease provides useful data that can provide support for research projects or an existing drug discovery program. How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained. Designing new treatments for rheumatic diseases, such as rheumatoid arthritis (RA), requires complex immunocompetent models that depend on intricate cytokine networks. Using local cytokines, signal transduction and transcription factor molecules as potential biomarkers to monitor disease and treatment efficacy is the best method to follow the progression of tissue damage and repair when testing an unknown compound or biologic. Described here in this report, a novel method for the non-enzymatic extraction and measurement of cytokines and signal transducers and activators of transcription (STAT) molecules using Luminex® bead array technology in two different mouse models for human RA--collagen antibody-dependent arthritis (CAIA) and collagen-induced arthritis (CIA).

Results: Dynamic expression of several pro-inflammatory cytokines responsible for promoting disease augmentation overtime were monitored, such as IL-1β, TNFα, IL-6 and IL-12, locally in the paws of affected animals directly ex vivo. Local cytokine responses could be matched with serum cytokine levels and joint pathology results. In addition, STAT1, 3, and 5a/b activation status could be monitored with confidence using specifically formulated extraction buffer that protected the phosphorylation site. STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone. Here reported a novel method of extracting joint fluid from the paws of inflamed mice coupled with powerful multiplex bead technology allowing us to measure cytokine responses, pharmacodynamic markers such as STATs and pharmacokinetic analysis of dosed agent all from the same sample directly ex vivo.

Conclusions: This method is powerful in that it is applicable to multiple autoimmunity model types, streamlines ex vivo readouts in a high-throughput manner, and allows multiplexing providing the investigator with an array of options and possible analytes when developing preclinical animal models to support drug discovery efforts in the search for new treatments for rheumatic diseases.

Show MeSH
Related in: MedlinePlus