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Reactivity of rat bone marrow-derived macrophages to neurotransmitter stimulation in the context of collagen II-induced arthritis.

Muschter D, Göttl C, Vogel M, Grifka J, Straub RH, Grässel S - Arthritis Res. Ther. (2015)

Bottom Line: Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA.Apoptosis remained unaffected.We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

View Article: PubMed Central - PubMed

Affiliation: Experimental Orthopedics, Centre for Medical Biotechnology, Biopark I, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany. dominique.muschter@klinik.uni-regensburg.de.

ABSTRACT

Introduction: Numerous observations indicate that rheumatoid arthritis (RA) has a bone marrow component. In parallel, local synovial changes depend on neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze whether collagen II-induced arthritis (CIA) has an impact on number, adhesion, apoptosis, and proliferation of the macrophage subset of bone marrow cells and how alterations in neurotransmitter microenvironment affect these properties.

Methods: Bone marrow-derived macrophages (BMMs) were isolated from Dark Agouti rats at different stages of CIA, and number, adhesion, caspase 3/7 activity, and proliferation were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA), and vasoactive intestinal peptide (VIP).

Results: Opposed to enhanced CD11b(+) (cluster of differentiation 11b-positive) and EMR1(+) (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1-positive) cells, characterizing the macrophage subset, in native bone marrow of rats with acute inflammatory arthritis, we found decreased numbers of CIA macrophages after enrichment and culture in comparison with healthy (control) animals. Adhesion studies revealed significantly reduced attachment to plastic in acute arthritis and collagen type I and fibronectin in chronic arthritis. Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA. Apoptosis remained unaffected. Neurotransmitter stimulation profoundly affected proliferation, adhesion, and apoptosis of BMMs from CIA and control rats, depending on disease time point. Cultured BMMs from CIA and control animals expressed neurotransmitter receptors for ACh, VIP and NA, but the expression profile seemed not to be affected by CIA.

Conclusions: Induction of CIA distinctly inhibits proliferation of BMMs in low- and non-inflammatory phases and reduces attachment to plastic at the acute inflammatory arthritis stage and adhesion to collagen I and fibronectin at the chronic stage. Influence of neurotransmitter stimulation on adhesion, apoptosis, and proliferation is altered by CIA depending on disease stage. We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

No MeSH data available.


Related in: MedlinePlus

Neurotransmitter receptor profile during course of CIA. Adrenergic receptors alpha 1D, alpha 2B, and beta 2 and muscarinic ACh receptor M5 and the alternative VIP receptor PACAP receptor 1 were stained in BMMs from control and CIA animals 20 (a) and 40 (b) days post immunization. Paraformaldehyde-fixed cells were stained after 5 days of differentiation in the presence of M-CSF and RANKL, and sections containing only BMMs were photographed. Nuclei were counterstained with DAPI. N = 4 (control and CIA). Magnification 200×. ACh acetylcholine, AChR acetylcholine receptor, BMM bone marrow-derived macrophage, CIA collagen II-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, M-CSF macrophage colony-stimulating factor, NA noradrenaline, PACAP pituitary adenylate cyclase-activating peptide, RANKL receptor activator of nuclear factor-kappa-B ligand, VIP vasoactive intestinal peptide
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Fig2: Neurotransmitter receptor profile during course of CIA. Adrenergic receptors alpha 1D, alpha 2B, and beta 2 and muscarinic ACh receptor M5 and the alternative VIP receptor PACAP receptor 1 were stained in BMMs from control and CIA animals 20 (a) and 40 (b) days post immunization. Paraformaldehyde-fixed cells were stained after 5 days of differentiation in the presence of M-CSF and RANKL, and sections containing only BMMs were photographed. Nuclei were counterstained with DAPI. N = 4 (control and CIA). Magnification 200×. ACh acetylcholine, AChR acetylcholine receptor, BMM bone marrow-derived macrophage, CIA collagen II-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, M-CSF macrophage colony-stimulating factor, NA noradrenaline, PACAP pituitary adenylate cyclase-activating peptide, RANKL receptor activator of nuclear factor-kappa-B ligand, VIP vasoactive intestinal peptide

Mentions: To assess whether the neurotransmitter reactivity of BMMs is altered during the time course of CIA, we analyzed expression of receptors for ACh, NA, and VIP. We detected mRNA expression of various neurotransmitter receptors (Additional file 3: Figure S2) and decided to further analyze protein expression of ARs α1D, α2B, and β2, PACAP receptor 1 (an alternative VIP receptor), and muscarinic ACh receptor M5 (Fig. 2). Receptors for immunostaining were selected upon searching the current literature for references indicating if any of the receptor subtypes showed altered expression or effector functions in physiological and pathophysiological conditions. Alpha2-ARs play a role in regulation of TNF release from macrophages and can be switched from pro-inflammatory TNF release in healthy conditions to inhibition of TNF release in a chronic constriction injury rat model [17]. This switch would be of interest, as blockade of TNF with therapeutic antibodies seems to play a pivotal role in restoring defective macrophage apoptosis observed in RA [18] or, as presented in a conflicting study, in inhibiting cell migration into inflamed synovial regions of RA patients [19]. Alpha1D-AR mRNA was detected in peripheral blood monocytes from patients with juvenile RA but not in peripheral blood mononuclear cells (PBMCs) of healthy donors [20], indicating a regulatory role for this receptor subtype under inflammatory conditions. For β2-AR, alterations in the sympathetic-to-immune cell signaling during adjuvant-induced arthritis, including immune organ-dependent changes in β2-AR expression, intracellular signaling, and receptor phosphorylation, have been described [21]. Protein analysis of PACAP receptor 1 was included because of its described anti-inflammatory actions representing a promising target for an anti-inflammatory therapy [22]. We detected mRNA for M1, M3, and M5 muscarinic ACh receptors and decided to include only M5 for immunostaining. M3 and M5, but not M1, mRNAs were expressed by PBMCs described by Costa et al. [23]. The rare subtype M5 showed opposite effects in two different studies, making it of specific interest in our study: transfected NIH3T3 cells showed a pro-proliferative response upon M5 receptor agonism [24], whereas a human melanoma cell line, A2058, displayed reduced clonogenic potential using a novel signaling pathway via M5 receptor stimulation [25].Fig. 2


Reactivity of rat bone marrow-derived macrophages to neurotransmitter stimulation in the context of collagen II-induced arthritis.

Muschter D, Göttl C, Vogel M, Grifka J, Straub RH, Grässel S - Arthritis Res. Ther. (2015)

Neurotransmitter receptor profile during course of CIA. Adrenergic receptors alpha 1D, alpha 2B, and beta 2 and muscarinic ACh receptor M5 and the alternative VIP receptor PACAP receptor 1 were stained in BMMs from control and CIA animals 20 (a) and 40 (b) days post immunization. Paraformaldehyde-fixed cells were stained after 5 days of differentiation in the presence of M-CSF and RANKL, and sections containing only BMMs were photographed. Nuclei were counterstained with DAPI. N = 4 (control and CIA). Magnification 200×. ACh acetylcholine, AChR acetylcholine receptor, BMM bone marrow-derived macrophage, CIA collagen II-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, M-CSF macrophage colony-stimulating factor, NA noradrenaline, PACAP pituitary adenylate cyclase-activating peptide, RANKL receptor activator of nuclear factor-kappa-B ligand, VIP vasoactive intestinal peptide
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496866&req=5

Fig2: Neurotransmitter receptor profile during course of CIA. Adrenergic receptors alpha 1D, alpha 2B, and beta 2 and muscarinic ACh receptor M5 and the alternative VIP receptor PACAP receptor 1 were stained in BMMs from control and CIA animals 20 (a) and 40 (b) days post immunization. Paraformaldehyde-fixed cells were stained after 5 days of differentiation in the presence of M-CSF and RANKL, and sections containing only BMMs were photographed. Nuclei were counterstained with DAPI. N = 4 (control and CIA). Magnification 200×. ACh acetylcholine, AChR acetylcholine receptor, BMM bone marrow-derived macrophage, CIA collagen II-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, M-CSF macrophage colony-stimulating factor, NA noradrenaline, PACAP pituitary adenylate cyclase-activating peptide, RANKL receptor activator of nuclear factor-kappa-B ligand, VIP vasoactive intestinal peptide
Mentions: To assess whether the neurotransmitter reactivity of BMMs is altered during the time course of CIA, we analyzed expression of receptors for ACh, NA, and VIP. We detected mRNA expression of various neurotransmitter receptors (Additional file 3: Figure S2) and decided to further analyze protein expression of ARs α1D, α2B, and β2, PACAP receptor 1 (an alternative VIP receptor), and muscarinic ACh receptor M5 (Fig. 2). Receptors for immunostaining were selected upon searching the current literature for references indicating if any of the receptor subtypes showed altered expression or effector functions in physiological and pathophysiological conditions. Alpha2-ARs play a role in regulation of TNF release from macrophages and can be switched from pro-inflammatory TNF release in healthy conditions to inhibition of TNF release in a chronic constriction injury rat model [17]. This switch would be of interest, as blockade of TNF with therapeutic antibodies seems to play a pivotal role in restoring defective macrophage apoptosis observed in RA [18] or, as presented in a conflicting study, in inhibiting cell migration into inflamed synovial regions of RA patients [19]. Alpha1D-AR mRNA was detected in peripheral blood monocytes from patients with juvenile RA but not in peripheral blood mononuclear cells (PBMCs) of healthy donors [20], indicating a regulatory role for this receptor subtype under inflammatory conditions. For β2-AR, alterations in the sympathetic-to-immune cell signaling during adjuvant-induced arthritis, including immune organ-dependent changes in β2-AR expression, intracellular signaling, and receptor phosphorylation, have been described [21]. Protein analysis of PACAP receptor 1 was included because of its described anti-inflammatory actions representing a promising target for an anti-inflammatory therapy [22]. We detected mRNA for M1, M3, and M5 muscarinic ACh receptors and decided to include only M5 for immunostaining. M3 and M5, but not M1, mRNAs were expressed by PBMCs described by Costa et al. [23]. The rare subtype M5 showed opposite effects in two different studies, making it of specific interest in our study: transfected NIH3T3 cells showed a pro-proliferative response upon M5 receptor agonism [24], whereas a human melanoma cell line, A2058, displayed reduced clonogenic potential using a novel signaling pathway via M5 receptor stimulation [25].Fig. 2

Bottom Line: Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA.Apoptosis remained unaffected.We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

View Article: PubMed Central - PubMed

Affiliation: Experimental Orthopedics, Centre for Medical Biotechnology, Biopark I, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany. dominique.muschter@klinik.uni-regensburg.de.

ABSTRACT

Introduction: Numerous observations indicate that rheumatoid arthritis (RA) has a bone marrow component. In parallel, local synovial changes depend on neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze whether collagen II-induced arthritis (CIA) has an impact on number, adhesion, apoptosis, and proliferation of the macrophage subset of bone marrow cells and how alterations in neurotransmitter microenvironment affect these properties.

Methods: Bone marrow-derived macrophages (BMMs) were isolated from Dark Agouti rats at different stages of CIA, and number, adhesion, caspase 3/7 activity, and proliferation were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA), and vasoactive intestinal peptide (VIP).

Results: Opposed to enhanced CD11b(+) (cluster of differentiation 11b-positive) and EMR1(+) (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1-positive) cells, characterizing the macrophage subset, in native bone marrow of rats with acute inflammatory arthritis, we found decreased numbers of CIA macrophages after enrichment and culture in comparison with healthy (control) animals. Adhesion studies revealed significantly reduced attachment to plastic in acute arthritis and collagen type I and fibronectin in chronic arthritis. Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA. Apoptosis remained unaffected. Neurotransmitter stimulation profoundly affected proliferation, adhesion, and apoptosis of BMMs from CIA and control rats, depending on disease time point. Cultured BMMs from CIA and control animals expressed neurotransmitter receptors for ACh, VIP and NA, but the expression profile seemed not to be affected by CIA.

Conclusions: Induction of CIA distinctly inhibits proliferation of BMMs in low- and non-inflammatory phases and reduces attachment to plastic at the acute inflammatory arthritis stage and adhesion to collagen I and fibronectin at the chronic stage. Influence of neurotransmitter stimulation on adhesion, apoptosis, and proliferation is altered by CIA depending on disease stage. We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

No MeSH data available.


Related in: MedlinePlus