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Direct action of immunoglobulin G on primary sensory neurons through Fc gamma receptor I¹

Department of Applied Pharmacology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

²Correspondence: Department of Applied Pharmacology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: kuraisiy{at}ms.toyama-mpu.ac.jp

SPECIFIC AIMS

Immunoglobulin G (IgG) binds specific antigens and IgG–antigen complexes primarily activate immune cells and a complement system. These immune reactions are known to affect neural functions through the production and release of mediators such as cytokines. The aim of this study was to determine the presence of IgG Fc receptors (FcRs) in the binding of IgG to and the action of IgG–antigen complex on the sensory neurons in mice.

PRINCIPAL FINDINGS

1. Expression of FcRI on mouse dorsal root ganglion neurons
Double immunostaining of mouse dorsal root ganglion (DRG) cells with antibodies against FcRI (CD64) and PGP9.5 (a general neuronal marker) revealed the expression of FcRI-like immunoreactivity in neurons (PGP9.5-positive cells) and glias (PGP9.5-negative cells) (Fig. 1 A). Western blot analysis confirmed the presence of FcRI, but not FcRII (CD32) and FcRIII (CD16), in the DRG neurons (Fig. 1B) . There were all types of FcR in the dermis and none in the sympathetic ganglion neurons.

View larger version (23K): [in this window] [in a new window]   Figure 1. Expression of FcRI in DRG neurons and glias. A) Immunocytochemical staining of cultured mouse DRG cells with anti-FcRI antibody (green) and anti-PGP9.5 antibody (red). Arrowheads and arrows indicate neurons and glias, respectively. Scale bar = 20 µm. B) Western blot analysis of FcRI, FcRII, and FcRIII in the mouse dermis, sympathetic ganglion (SG) neurons, and DRG neurons. Two sets of three samples (dermis, SG, and DRG) and molecular markers were electrophoresed on a gel and transferred to a membrane. The membrane was cut into 2 sheets; one was reacted with anti-FcRI antibody and another with anti-FcRII/FcRIII antibody. C) Frequency distribution of FcRI-positive neurons in the DRG. Hatched columns represent FcRI-positive neurons (n=505), open columns FcRI-negative neurons (n=483). The inset shows examples of FcRI immunoreactive DRG neurons. Scale bar = 20 µm.

The frequency distribution of FcRI immunoreactive neurons is shown in Fig. 1C . When 988 cultured DRG neurons were examined, 505 neurons (51%) were immunoreactive for FcRI. Four hundred and forty-six (57%) of 778 small-sized neurons (15 µm in diameter), 44 (46%) of 96 medium-sized neurons (>15 and 25 µm), and 15 (13%) of 114 large-sized neurons were immunoreactive for FcRI. Eighty-eight percent of 505 FcRI immunoreactive neurons were small in size.

2. Binding of IgG to DRG neurons and nerve fibers in the skin
An intradermal injection of ragweed pollen significantly elicited scratching of the injected site in ragweed pollen-sensitized mice. IgG was purified from the sera of the ragweed pollen-sensitized and nonsensitized mice. Cultured DRG neurons were treated with total IgG purified from ragweed pollen-sensitized and nonsensitized mice, then with FITC-labeled ragweed pollen. When treated with IgG from ragweed pollen-sensitized mice, DRG neurons were labeled with IgG and ragweed pollen. Treatment with IgG from nonsensitized mice resulted in the binding of IgG, but not ragweed pollen, to the neurons.

There were many PGP9.5 immunoreactive nerve fibers in the dermis just under the epidermal basal layer. After intradermal injection of FITC-labeled ragweed pollen into the nonimmunized mouse, there were no ragweed pollen signals in the skin adjacent to injection site. On the other hand, there were many ragweed pollen signals in the corresponding skin region, and the majority of ragweed pollen was on the nerve fibers.

3. Increase of intracellular Ca²⁺ concentration induced by antigen–IgG immune complex in DRG neurons
Administration of ragweed pollen (5 ng) did not affect the concentration of intracellular Ca²⁺ ions in cultured DRG neurons untreated or treated with nonspecific IgG (10 ng). Although administration of total IgG (10 ng) from ragweed pollen-immunized mice did not affect intracellular Ca²⁺ concentration, ragweed pollen (5 ng) gradually increased it in pretreated DRG neurons. This increase was completely suppressed by anti-FcRI antibody (10 ng) and by removing extracellular Ca²⁺ ions. It was markedly suppressed by the voltage-dependent L-type Ca²⁺ channel blocker nicardipine (5 µM) and the voltage-dependent N-type Ca²⁺ channel blocker -conotoxin GVIA (1 µM).

4. Release of substance P induced by antigen–IgG immune complex from DRG neurons
Although both anti-ragweed pollen IgG (10 ng) alone and ragweed pollen (5 ng) alone were without effect, ragweed pollen (5 ng) released substance P from cultured DRG neurons (1x10⁶ cells) pretreated with anti-ragweed pollen IgG, but not with nonspecific IgG, at a dose of 10 ng (Fig. 2 ). This release was markedly inhibited by anti-FcRI antibody (10 ng) and L-type Ca²⁺ channel blockers calcisepine (0.3 µM) and nicardipine (5 µM) (Fig. 2) . It was almost abolished by the N-type Ca²⁺ channel blockers -conotoxin GVIA (1 µM) and -conotoxin MVIIA (1 µM) (Fig. 2) . High K⁺ (20 mM) stimulation induced substance P release, the amount of which was similar to that of RP-IgG complex stimulation.

View larger version (34K): [in this window] [in a new window]   Figure 2. Ragweed pollen (RP) releases substance P from anti-RP IgG-pretreated DRG neurons. The concentration of substance P in the medium of primary cultures of mouse DRG neurons was determined 10 min after RP (5 ng) administration or 20 mM KCl stimulation. Anti-FcRI antibody (10 ng) was administered 30 min before RP administration, total IgG (10 ng) containing anti-RP IgG, nonspecific IgG (10 ng), and Ca2+ channel blockers 10 min before RP administration. The concentration of Ca2+ channel blockers was as follows: calcisepine, 0.3 µM; nicardipine, 5 µM; -conotoxin (-CoTx) GVIA, 1 µM; -CoTx MVIIA, 1 µM. Data are means ± SE (n=6). *P < 0.05 vs. nonspecific IgG; #P < 0.05 vs. anti-RP IgG (Dunnett’s multiple comparisons).

CONCLUSIONS AND SIGNIFICANCE

IgG–antigen immune complex has been known to activate immune cells such as macrophages and mast cells to release mediators (i.e., cytokines and amines) that act on neurons. Thus, IgG–antigen immune complex is considered to indirectly affect neuronal functions. The present study show that the high-affinity IgG receptor FcRI is expressed in primary sensory neurons and that IgG–antigen immune complex is formed on the sensory neurons to affect their functions.

IgG–antigen complex increased intracellular Ca²⁺ concentration in and the release of substance P from primary sensory neurons, which was markedly suppressed by voltage-dependent L- and N-type Ca²⁺ channel blockers. The results suggest that IgG–antigen complex depolarizes the sensory neurons.

Small-sized DRG neurons have chiefly unmyelinated C-fibers, the majority of which belong to nociceptors, and C fibers play an important role in pain, itch, and flare. Therefore, that FcRI was expressed mainly in small-sized neurons raises the possibility that the action of IgG–antigen immune complex on sensory neurons produces sensations such as pain and itch and inflammatory responses.

The direct immunoglobulin–neuron linkage may be a new target for the development of new drug for immune diseases.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic representation of the action of antigen–IgG immune complex on the sensory neuron. Binding of antigen (Ag) to IgG bound to FcRI may depolarize the sensory neuron to activate voltage-dependent L- and N-type Ca2+ channels. This results in release of neurotransmitters such as substance P (SP) and may cause sensory signals that are conducted to the central nervous system.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1169fje

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