Antibodies against gonadotropin-releasing hormone (GnRH) and destruction of enteric neurons in 3 patients suffering from gastrointestinal dysfunction

Abstract

Background: Antibodies against gonadotropin-releasing hormone (GnRH) and gastrointestinal dysmotility have been found after treatment with GnRH analogues. The aim of this study was to examine the presence of such antibodies in patients with dysmotility not subjected to GnRH treatment and study the anti-GnRH antibody effect on enteric neurons viability in vitro.

Methods: Plasma and sera from 3 patients suffering from either enteric dysmotility, irritable bowel syndrome (IBS) or gastroparesis were analysed for C-reactive protein (CRP), and for GnRH antibodies and soluble CD40 by ELISA methods. Primary cultures of small intestinal myenteric neurons were prepared from rats. Neuronal survival was determined after the addition of sera either from the patients with dysmotility, from healthy blood donors, antiserum raised against GnRH or the GnRH analogue buserelin. Only for case 1 a full-thickness bowel wall biopsy was available for immunohistochemical analysis.

Results: All 3 patients expressed antibodies against GnRH. The antibody titer correlated to the levels of CD40 (rs = 1.000, p < 0.01), but not to CRP. Serum from case 3 with highest anti-GnRH antibody titer, and serum concentrations of sCD40 and CRP, when added to cultured rat myenteric neurons caused remarkable cell death. In contrast, serum from cases 1 and 2 having lower anti-GnRH antibody titer and lower sCD40 levels had no significant effect. Importantly, commercial antibodies against GnRH showed no effect on neuron viability whereas buserelin exerted a protective effect. The full-thickness biopsy from the bowel wall of case 1 showed ganglioneuritis and decrease of GnRH and GnRH receptor.

Conclusion: Autoantibodies against GnRH can be detected independently on treatment of GnRH analogue. Whether the generation of the antibody is directly linked to neuron degeneration and chronic gastrointestinal symptoms in patients with intestinal dysmotility, remains to be answered.

Figure 1

Control myenteric ganglia. A: one GnRH positive neuron (thick arrow) and two GnRH negative neurons within the ganglion. (GnRH immunohistochemistry; bar: 20 μm). B: three GnRH receptor positive neurons (thin arrows) and three receptor negative nerve cells (thick arrows) in control ganglion. (GnRH receptor immunohistochemistry; bar: 20 μm).

Figure 2

The correlation between the concentration of antibodies against gonadotropin releasing hormone (GnRH) at 1:40 dilution and soluble CD40 in serum (pg/ml), r_s = 1.000, p < 0.01. The measured absorbance at 405 nm of different plasma samples using the ELISA procedure described was transformed to the corresponding rabbit anti-GnRH with the aid of the standard curve. The sample was considered as positive if the absorbance was > 0.800.

Figure 3

Immunocytochemical staining with PGP of myenteric neurons grown for 6 days. The cultured neurons survive well; they group into ganglion-like structures and grow a prominent arborising network of nerve terminals. Bar 40 μm.

Figure 4

Neuronal survival of neurons pre-cultured for 4 days followed by 2 days of culture in the presence of (A) serum from case 3 (n = 5), (B) serum from case 1 (n = 8), (C) serum from case 2 (n = 3), (D) serum from healthy blood donors (n = 8), (E) gonadotropin-releasing hormone (GnRH) antibodies raised in rabbit (n = 6-7) and (F) buserelin (n = 8). Addition of sera from cases number one and two, from healthy blood donors or GnRH antibodies caused no change in neuronal survival while serum from case 3 significantly and concentration-dependently reduced neuronal survival. Buserelin markedly enhanced neuronal survival in a concentration-dependent manner. *** p < 0.01, as compared to the controls run in parallel.

Figure 5

Details of a myenteric ganglion and surrounding muscle tissue. 5A. The cytoplasm of the enlarged neuron is filled with small tightly packed eosinophilic granules, the nucleus is compressed, elongated (large arrow). There are two other neurons with vacuolized cytoplasm (double arrows) and another small, shrunken neuron (short arrow) (Haematoxylin & eosin;bar: 20 μm). 5B. Eosinophilic residual body with shadows of vacuoles of a necrotic neuron (large arrow). The double arrows show a degenerating neuron containing very large nucleolus and extremely pale cytoplasm with a few perinuclear vacuoles. (Haematoxylin & eosin; bar: 20 μm). 5C. Ten μm large, activated T-lymphocytes in the vicinity of two neurons (arrows). (CD3 immunohistochemistry; bar: 20 μm). 5D and E show detail of the circular muscle with a small nerve as followed up on serial sections. 5D. There is a small cell containing a densely stained rounded nucleus (arrow) along the small nerve. (Neurofilament immunohistochemistry; bar: 20 μm). 5E. Several CD3+ T-lymphocytes are seen along the nerve shown on 5D. (CD3 immunohistochemistry; bar: 20 μm).