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γ-aminobutyric acid-transaminase activity in the human thymus after administration of interferons
␥-Aminobutyric Acid-Transaminase Activity in the Human Thymus After Administration of Interferons Daniela Cavallotti, Marco Artico, Vito D’Andrea, and Carlo Cavallotti ABSTRACT: The purpose of this article is to study the amounts of ␥-aminobutyric acid-transaminase (GABA-t) during immune response in the human thymus. GABA-t was studied by biochemical and histochemical methods in 7 immunostimulated (treated) and 7 non-immunostimulated (untreated) patients (4 young adult, age range: 24 –36 years; 3 older adult, age range: 56 – 66 years). Immunostimulation was performed using interferon drugs for 6 months. After the histoenzymatic staining of GABA-t activity, the slides containing the samples of thymus of treated and untreated patients underwent quantitative analysis of images. The present results provide direct evidence that the immune response increases the level of GABA-t contained in vessels, parenchyma and
ABBREVIATIONS GABA-t ␥-aminobutyric acid-transaminase MS multiple sclerosis BSA bovine serum albumin IL interleukin QAI quantitative analysis of images NAD nicotinamide adenine dinucleotide
INTRODUCTION Numerous experimental studies indicate that a link exists between the immune and neuroendocrine systems in mammals, including humans [1]. In both the primary and secondary lymphoid organs of rats, the norepinephrine amount is altered after an immune stimulating
From the 2nd Neurologic Clinic (D.C.), 3rd Surgical Clinic (V.D.), Department of Cardiovascular and Respiratory Sciences (C.C.), and Department of Pharmacology of Natural Molecules and General Physiology (M.A.), University “La Sapienza”, Rome, Italy. Address reprint requests to: Carlo Cavallotti, M.D., Department of Cardiovascular and Respiratory Sciences, University of Roma “La Sapienza”—Via A. Borelli, 50 00161 Rome, Italy; Tel: ⫹39 06 4958291; Fax: ⫹39 06 4957669; E-Mail: [email protected]. Received November 17, 1999; revised March 28, 2000; accepted March 30, 2000. Human Immunology 61, 697–704 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.
nerve fibers of the thymus. Treatment with interferon is also capable of increasing the protein content of the thymus. The biochemical data together with the histoenzymatic results provide evidence for a localization of GABA-t in the thymic gland. Moreover, ␥-aminobutyric acid can be considered as one of the linking molecules between the immune and nervous functions of the human thymus. Human Immunology 61, 697–704 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: ␥-aminobutyric acid-transaminase (GABA-t); immune response; thymus; interferon; quantitative analysis of images (QAI)
CU NADH IFNs IFN-
conventional units nicotinamide adenine dinucleotide hydrogenated interferons interferon-beta
therapy [2]. On the contrary, in immunodeficient mice some of the neurotransmitters of the thymus present numerous changes [3]. Weihe and coworkers [4], studying the molecular anatomy of the neuro-immune connection in a variety of mammals, demonstrated that numerous neuropeptides play a role as linking molecules between neuro-immune and neuro-endocrine functions of the thymic gland. ␥-Aminobutyric acid (GABA) can be considered as one of the linking molecules between the neural and neuroendocrine component of the human thymus [5]. GABA is widely recognized as a major inhibitory neurotransmitter in the vasculature of vertebrates [6]. GABA, its related enzymes, i.e., glutamate decarboxylase and ␥-aminobutyric acid-transaminase (GABA-t), as well as GABA 0198-8859/00/$–see front matter PII S0198-8859(00)00130-0
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receptors, have been demonstrated in a wide variety of peripheral tissues [7, 8]. Although the physiological relevance of GABA in most of the peripheral tissues remains to be clarified, there are convincing lines of evidence to suggest a role for GABA as an inhibitory neurotransmitter in the vertebrate peripheral nervous system [9]. In fact, GABA is present in the mouse thymus and undergoes a significant increase during the immune response, i.e., following challenge with a T-cell dependent antigen [10]. GABA-t activity in thymus of rats and mice has been demonstrated in our laboratories many years ago [11]. GABA-t is the enzyme that catabolizes GABA, a neurotransmitter that increases up to elevated levels in the thymus during an immune response [10]. In a recent article, we have demonstrated the occurrence of GABA-t also in nerve fibers of thymus from young and elderly men [12]. The purpose of this article is to study whether the increased levels of GABA found in the mouse thymus during the immune response [10] are confirmed by an increase of GABA-t in the human thymus after an immune-response. Moreover, the relationships between GABA-t and interferon-beta ( IFN-) in the human thymus in immunostimulated or in non-immunostimulated patients have been analyzed with special reference to the effects of immunomodulatory molecules interferons (IFNs) on neurotransmitter molecules (GABA). In fact, an increase in GABA-t content and the presence of IFN 1 may support the hypothesis that immunomodulatory molecules are able to exert some effects on neurotransmitters, thus providing reasonable evidence for a link between both systems. MATERIALS AND METHODS The experimental procedures performed in this study include: (1) patient’s immune stimulation, (2) thymic biopsies, (3) histochemical demonstration of GABA-t, (4) estimation of protein content, (5) biochemical dosage of GABA-t, (6) quantitative analysis of images, (7) statistical analysis of data. Each procedure is briefly explained. Patient’s Immune Stimulation In our city, many neurological hospitals are authorized as national data points for the study of multiple sclerosis (MS). In these hospitals, patients with MS are enrolled and treated by IFNs. -1a interferon (Avonex, SolvayDuphar, Olst, The Netherlands) belongs to a group of drugs that modulate the regulation of the human immune system. -1b interferon (Betaferon, Schering AG, Germany) also modifies the response of the human immune system. Recently, human recombinant  interferon (Serobif, Serono SPA, Italy) has been introduced
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into clinical practice as an immunomodulating systemic agent. All these mentioned interferons can be used in the treatment of patients affected by recurrent-remittent MS, whose clinical history is marked by at least two episodes of neurological involvement during the previous two years. In order to study the possible involvement of the thymus gland in MS, a number of patients with MS or affected by other neuroimmunological diseases (excluding myasthenia gravis), treated or untreated with interferon drugs, underwent (for diagnostic reasons) a surgical biopsy of the thymus. Small fragments of these thymic biopsies drawn from immunostimulated patients affected by MS or by other neurological diseases were thus available for our experiments. Our studies were approved by the Ethical Committees of the involved hospitals and patients also gave their informed written consent. In National Clinical Centers the following therapeutic procedures were used for administration of interferon drugs: 36 patients (age range 24 – 66 years) were randomly divided into three groups and treated by three different protocols in order to obtain a remission of their illness. Group 1: 14 patients enrolled; 6 of them treated for at least 6 months by one intramuscular injection of Avonex ( 1a interferon) weekly. Group 2: 12 patients enrolled; 5 of them treated for at least 6 months by three intradermal injections of Betaferon ( 1b interferon) 8.000.000 of International Units (IU) weekly. Group 3: 10 patients enrolled; 5 of them treated for at least 6 months (the drug is still completing the official registration course required by the Italian Health Authorities) by three intradermal or intramuscular injections of Serobif (Human recombinant) (-interferon) corresponding to 6.000.000 of IU weekly. Thymic Biopsies Three patients of the first group, two patients of the second group, and two patients of the third (all treated with interferons, n ⫽ 16) underwent surgical thymic biopsies. The remaining group of patients (n ⫽ 20) with MS was not treated by interferons because their symptoms did not conform to the protocols approved by Health Ministry for treatment by means of interferons. Seven patients affected by MS and untreated with interferons or affected by other neurological diseases also underwent diagnostic thymic biopsy. Samples from these patients were considered as untreated and served as the control group. Small fragments of all these biopsies were carried quickly under dry ice to our laboratories where they were submitted to the experimental procedures. Table 1 con-
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TABLE 1 Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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Relevant characteristics of 14 patients enrolled in our medical protocols
Treatment Avonex Avonex Avonex Betaferon Betaferon Serobif Serobif None None None None None None None
Age (yrs)
IgM (mg/dl ⫾ S.E.M.)
56 66 24 60 30 32 28 25 58 32 60 36 64 27
220 ⫾ 1.8 198 ⫾ 1.3 232 ⫾ 1.5 241 ⫾ 1.9 196 ⫾ 2.1 210 ⫾ 1.8 224 ⫾ 1.5 110 ⫾ 1.3 96 ⫾ 0.8 162 ⫾ 1.2 124 ⫾ 1.1 150 ⫾ 1.3 136 ⫾ 1.5 120 ⫾ 1.0
Diagnosis a
MS MSa MSa MSa MSa MSa MSa MS w.t.b MS w.t.b MS w.t.b MS w.t.b MS w.t.b MS w.t.b MS w.t.b
All patients underwent diagnostic thymic biopsy. Other 22 patients didn’t undergo thymic biopsy and so were excluded from the present experiments. IgM were tested many folds before the treatment and after 3, 4, 5, and 6 months from the beginning of therapy. The listed results are the mean values of four experiments (five for untreated patients) ⫾ SEM. The values before the treatment were excluded by treated patients, while were included for untreated patients. a MS ⫽ multiple sclerosis under treatment with -interferon owing to the clinical situation, as reported in Methods. b MS w.t. ⫽ multiple sclerosis without treatment.
tains all relevant characteristics of the patients enrolled in our medical protocols. Histochemical Demonstration of GABA-t Samples of human thymus coming from surgical biopsies in untreated or interferon-treated patients were rapidly placed in a dry-ice mixture for the histochemical demonstration of GABA-t activity. The Van Gelder method (1965) was used for histochemical detection of specific GABA-t [13], carrying out the slight modifications described in a previous paper of us to minimize enzyme diffusion [14]. In brief, each slice of thymus was stretched on preweighed slides, which were then newly weighed to estimate the weight of the sample. Samples were treated by histoenzymatic staining that involves the reduction of tetrazolium salts. Control sections were incubated without substrates or using, as inhibitors, 50 M of amino-oxalacetic acid or 50 M of Gabaculline or 200 M of acetylenic GABA or 100 M of ethanolamine sulfate, one at a time or simultaneously. Estimation of Protein Content In this group of experiments, the thymic biopsies were weighed and placed on dry ice, (specimens for GABA-t histochemical staining) or into an ice-cold homogenisation buffer (samples for estimating the protein content and the GABA-t activity).
Tissue protein concentrations were determined by the method described by Lowry et al. [15] using bovine serum albumin (BSA) as standard. Biochemical Dosage of GABA-t Biochemical dosage of GABA-t activity (EC 2.6.1.19 4-aminobutyrate 2-oxoglutarate aminotransferase) was carried out according to the method of Jung et al. [16] using 6 M GABA plus 5 M 2-oxoglutarate as substrate and determining the formation of NADH starting by NAD. Specific GABA-t activity was calculated by subtracting the blank values (measured in the presence of 50 M of amino-oxalacetic acid as inhibitor or in presence of other inhibitors or in absence of substrate) from the total activity. Other GABA-t inhibitors used were Gabaculline 50 M, acetylenic GABA 200 M or ethanolamine-o-sulfate 100 M. The biochemical values obtained using all these inhibitors (one at a time or simultaneously) can be considered as blank values and must be subtracted from the values of total activity. Quantitative Analysis of Images In order to evaluate the effects of immune-stimulation on GABA-t levels, a quantitative analysis of the intensity of the histochemical staining was performed on slides by means of a Quantimet Analyzer (Leica Microsystems Imaging Solutions, Cambridge, UK). The values of control sections, incubated without substrates, were considered as “zero.” Examinations were performed separately for each type of treatment on 7 treated patients and on 7 untreated patients, on at least fifteen slices for each patient, evaluating the standard error of the mean (⫾SEM). Final values must be submitted to statistical data analysis. The values reported represent the intensity of staining for each type of treatment and are expressed in conventional units (C.U.) ⫾SEM. Conventional units are arbitrary units that are furnished directly by the display of the Quantimet Analyzer [16]. Statistical Analysis of Data The statistical methods used throughout this study must be interpreted as an accurate description of the data rather than a statistical inference of such data. The preliminary studies of each value were performed with the of basic sample statistics. Mean values, maximum and minimum limits, standard deviation (SD), standard error of the mean (SEM) and correlation coefficients were carried out according to Serio [17]. The relationship between each pair of variables was studied using the respective correlation coefficients grouped in a
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FIGURE 1 GABA-transaminase activity in the human thymus of a young man (27 years old) untreated with interferons (Magnification 250⫻). Due to the thymic involution the picture shows (arrow) a small adipose tissue infiltration. The wall of many lymphatic vessels (L) shows a positive reaction. Numerous reticular cells (R) also show a positive reaction. Many structures resembling nerve fibers (n) also show a positive reaction. Medullary zone on the right side, cortical zone on the left side.
correlation matrix, thus enabling us to study the existence of a linear (values next ⫹1 or ⫺1) or non-linear (values next 0⫹) dependency. Finally, a correlative analysis of the morphological and biochemical data was performed by comparing the significant differences for each age with the corresponding values of the other homogeneous groups. Correlation coefficients denote a significant level less than 0.001 (p ⬍ 0.001) while the correlation coefficient is not significant when p ⬎ 0.05 (n.s.). This correlation coefficient was calculated according to Castino and Roletto [18]. RESULTS Our results are reported in Figures 1 through 4 and summarized in Tables 1 through 4. Figure 1 shows that in an untreated young man (27 years old) the GABA-t activity is localized in the wall of lymphatic vessels (L), in numerous reticular cells (R) and in many structures FIGURE 2 GABA-transaminase activity in the human thymus of an old man (60 years of age) untreated with interferons (Magnification 250⫻). We can observe an evident adipose tissue infiltration (arrows). The wall of two venules (V) and of two lymphatic vessels (L) shows a positive reaction. A Hassall’s corpuscle (H), considered as expression of thymic involution, shows a negative reaction, while many reticular cells (R) show only a few positive reactions. Many structures resembling nerve fibers (arrows) show a positive reaction. Medullary zone on the right side, cortical zone on the left side.
resembling nerve fibers (n). Thymic tissue shows a small adipose tissue infiltration (arrow). Figure 2 depicts an untreated old man (60 years of age) only two lymphatic vessels (L) and two veins (V) show a positive staining for the GABA-t activity. A corpuscle of Hassall (H) appears unstained. The reticular cells also show a positive reaction. Moreover, we can observe an evident adipose tissue infiltration (arrow). Figure 3, which must be compared with Figure 1, is a sample from a young patient (28 years of age) previously treated with interferon drugs. This photograph shows a strong positive reaction for GABA-t activity localized in reticular cells (R) and in the wall of an arteriola (A), of a venula (V) and of numerous lymphatic vessels (L). In Figure 4, we can observe strong positive enzymatic staining in reticular cells (R) in many structures resembling nerve fibers (n) and in the wall of an arteriola (A) and of a venula (V). The samples of thymus were taken
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FIGURE 3 GABA-transaminase activity in the human thymus of a young patient (28 years of age) treated with interferons (Magnification 250⫻). We can observe the signs of a thymic involution (arrow). The wall of an arteriola (A), of a venula (V), of numerous lymphatic vessels (L) and the reticular cells (R) show a positive reaction. Medullary zone on the right side, cortical zone on the left side.
from an old man (age 66 years) previously treated with interferon drugs. By comparing Figure 2 with Figure 4, it is evident that interferon treatment induced an increase of GABA-t levels. Table 1 deals with the relevant characteristics of 14 patients (which underwent diagnostic thymic biopsy) enrolled in our medical protocols. As can be seen treatment with INF- induces an immune response that can be verified by serum elevated IgM. Table 2 displays the results of protein content in homogenates of thymus in adult or old patients, either treated or untreated with interferon. As can be seen, therapeutic treatment with interferon drugs modifies the protein content of thymic homogenates both in adult and in older patients. In fact, in untreated adult patients protein levels expressed as mg/g fresh weight of thymus are 15.8 mg while in treated adult patients this level goes up to 19.6 mg with a highly positive treated versus untreated significance index (p ⬍ 0.001). Moreover, in FIGURE 4 GABA-transaminase activity in the human thymus of an old patient (66 years of age) treated with interferons (Magnification 250⫻). We can observe a strong positive reaction in reticular cells (R) while a Hassall’s corpuscle (H) shows a negative reaction. Structures resembling nerve fibers (n) show an evident positive reaction. Moreover, we can observe the wall of an arteriola (A) and of a venula (V) showing a positive reaction. Medullary zone on the right side, cortical zone on the left side.
untreated older patients, the thymus protein content is 10.4 mg/g tissue fresh weight. It goes up to 12.8 mg/g tissue fresh weight in thymus of patients treated with interferon drugs, with a positive index of significance despite the limited number of samples examined (3 treated and 3 untreated). Table 3 deals with the values of biochemical dosages of GABA-t activity in the thymus of adult and older patients treated and untreated with interferon. Results are expressed as nM /mg protein/h. In untreated men (n ⫽ 4, i.e., four subjects) the mean value was 45.6. It rose to 93.2 in patients treated with interferon. In untreated older men (n ⫽ 3, i.e., three subjects) the mean value was 22.4. It rose to 39.7 in patients treated with interferon. By comparing the significant differences between treated and untreated patients we can see that the significance index is highly positive p ⬍ 0.001. Table 4 shows the results of quantitative image anal-
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TABLE 2
Thymus Adult patients Age 24–36 n⫽4 Older patients Age 56–66 n⫽3
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Protein content in homogenates of thymus of adult and older patients treated and untreated with interferon Untreated (n ⫽ 7) 15.8 ⫾ 0.19 10.4 ⫾ 0.13
Treated with interferona (n ⫽ 7) 19.6 ⫾ 0.22b 12.8 ⫾ 0.18b
Results are expressed as mg/g of fresh weight tissue. Each value is the mean value ⫾ SD of independent determinations from n. patients, carried out in triplicate. n ⫽ number of subjects a Treatment of patients with interferon drugs was performed by one of the three procedures reported in Methods, alternatively. b p ⫽ ⬍ 0.001 treated versus untreated.
ysis of the GABA-t levels in biopsies of the thymus of adult and old patients in treated or untreated subjects. The mean value in four adult untreated patients was 28.9 C.U. It rose to 58.3 in four adult treated patients. In three untreated older patients the mean value of GABA-t levels was 13.6 C.U., rising to 26.4 C.U. in patients of the same age treated with interferon. These results show that after therapeutic treatment with interferon the level of GABA-t is increased. The biochemical data together with the histoenzymatical results provide evidence for a localization of GABA-t in the thymic gland. Moreover, the administration of interferon modifies the levels of GABA-t.
TABLE 3
Thymus Adult patients Age 24–36 n⫽4 Older patients Age 56–66 n⫽3
TABLE 4
Biochemical dosage of GABA-transaminase activity in thymus of adult and older patients treated and untreated with interferon Untreated (n ⫽ 7)
Treated with interferona (n ⫽ 7)
45.6 ⫾ 3.12
93.2 ⫾ 4.36b
22.4 ⫾ 1.63
39.7 ⫾ 2.84b
Results are expressed as nM/mg protein/h. Each value is the mean value ⫾ SD of independent determinations from n. patients, carried out in triplicate. n ⫽ number of subjects a Treatment of patients with interferon drugs was performed by one of the three procedures reported in Methods, alternatively. b p ⫽ ⬍ 0.001 treated versus untreated.
Thymus Adult patients Age 24–36 n⫽4 Older patients Age 56–66 n⫽3
Quantitative analysis of images of the GABA-transaminase levels in thymic biopsies of adult and older patients treated and untreated with interferon after histoenzymatical staining Untreated (n ⫽ 7)
Treated with interferona (n ⫽ 7)
28.9 ⫾ 2.6
58.3 ⫾ 3.1b
13.6 ⫾ 1.4
26.4 ⫾ 2.3b
Results are expressed in Conventional Units (C.U.) (see Methods) ⫾ SEM. The analyzer was calibrated considering “zero” the values of control sections incubated without substrate or in presence of specific inhibitors. For other precautions used see methods. p was calculated comparing the significant differences between treated and untreated patients. n ⫽ number of subjects a Treatment of patients with interferon drugs was performed by one of the three procedures reported in Methods, alternatively. b p ⫽ ⬍ 0.001 treated versus untreated.
DISCUSSION There are alternative histological methods for visualizing GABA-containing elements in mammalian tissues. It was generally assumed that radiolabeled GABA, taken up by a particular tissue, specifically labels the GABAcontaining cells that can be visualized by autoradiography . Recent findings have revealed, however, that the GABA-accumulating cells do not necessarily correspond to the GABA-containing and GABA synthesizing cells [19 –21]. Another approach is the immunocytochemical localization of glutamate decarboxylase, the marker enzyme of GABA containing structures. This procedure, however, also has some disadvantages. The topography of glutamate decarboxylase within a cell does not necessarily match the localization of the small, rapidly diffusing, GABA molecule: additionally, there are marked species and tissue differences in the immunochemical properties of the enzymes of different origin [22]. In addition, the distribution of the catabolic enzyme GABA,-t may not reflect the location of GABA within a particular tissue, but the enzyme-substrate reaction, generally used for visualization of GABA-t [13, 23], can be universally applied for different species and tissues. However, the direct GABA immunocytochemical method is capable of demonstrating relatively low tissue concentrations, is highly specific, and can be used both for light and electron-microscopic samples. Moreover, there is no problem with species and tissue specificity [24, 25]. Taking into account the advantages and disadvantages of the above procedures, the GABA-t histoenzymatic
GABA-t in Thymus and Interferons Treatment
method developed by Van Gelder [13] was adapted and used for studying the distribution of GABA-t in human thymus in normal conditions and after interferon administration. A low marginal GABA-t activity was found in association with thymic blood vessels. The enzymatic activity was higher in the thymic biopsies coming from treated subjects. In one of our recent publications [12], we demonstrated that GABA-t content appears to be increased with age. On the contrary, in this study a decrease of GABA-t in thymuses of old patients (if compared to young patients) is demonstrated. This apparent discrepancy may be explained because in these mentioned study, we used thymic fragments harvested from autopsies and considering the whole thymus, while, in the present study, specimens were drawn from surgical biopsies, obtaining very small fragments confined to the thymic microenvironment only. In fact, in this study we excluded other thymic structures (different from the parenchymal microenvironment) in which GABA-t is widely distributed. In contrast to IFN-␥, which is primary immunomodulator (with little anti-viral activity) INFs- are considered to be primarily anti-viral agents with some immunomodulatory activity. Nevertheless, the role of INF- in the modulation of the immune response in MS is well documented [26]. Moreover, antiviral activity of -interferon seems to exert a useful effect in the therapy of recurrent MS [27]. However, National Health Authorities recommend the use of -interferons in similar experimental protocols and so we are unable to modify these procedures. Treatment with interferon drugs induces an immune response. This response can be verified with dosages of serum elevated antibody titers (see Table 1) and by means of common laboratory tests performed during hospitalisation (protein immuno-electrophoresis a.s.o.). A significant rise in thymic GABA-t levels was found after treatment with interferon drugs. The potential biological significance of this increase can be considered. GABA-t activity catabolizes the GABA and was, therefore, strictly correlated with GABA metabolism. Since GABA is able to function as an inhibitory transmitter at the level of cholinergic receptors on the neuromuscular junction, it is possible that it might function in the same manner on the cholinergic receptors of the thymic epithelial cells that synthesize the thymosin. Moreover, large amounts of GABA have been found incorporated in many thymosin peptides that have been sequenced in their amino-acidic composition. In conclusion, our results demonstrate that patients treated with interferon, a potent promoter of immune responses, show high levels of GABA-t activity in thymic structures (vessels, nerve fibers and parenchyma) in
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comparison to untreated patients. Therefore, at present, the significance of the enhancing effect of INFs- on the presence of GABA-t remains very uncertain. Treatment with INF is also capable of increasing the protein content of the thymus. In fact, our data show that in vivo INF- treatment doubles the intrathymic amounts of GABA-t in humans. Is this effect thymusspecific? GABA-t is specifically enhanced in the wall of blood vessels in different vascular districts including cerebral vessels. Because we did not study other tissues, we are unable to exclude if the effects of interferon- are thymus-specific or not. What is the meaning of intrathymic expression of GABA-t? As previously mentioned, GABA is a neurotransmitter that plays a role in many central and peripheral nerve fibers as well as an amino-acid with several functions in protein metabolism. GABA-ergic systems in the thymus undergo changes after immunostimulating therapy. In our laboratory, experiments are currently under way to study variations of other neurochemical parameters (density of receptor binding sites, affinity of receptors to specific ligands, turnover of these substances). The related results may provide more valuable information about the meaning of intrathymic expression of GABA-t. Our biochemical data, together with histoenzymatic qualitative and quantitative results, provide direct evidence for a specific localization of GABA-t in the thymic gland. Moreover, reticular cells, epithelial cells and structures resembling nerve fibers of the thymus show a positive GABA-t staining. ACKNOWLEDGMENTS
The present study was supported by grants from MURST, CNR and University of Rome “La Sapienza” (COD-DIP 98.043.14). The authors are greatly indebted to Drs. Cristoforo Morocutti and Ricardo Miledi for their suggestions and criticisms. The Medlines, Internet and other informatic consulting services of Drs. M. Cameroni, F. M. Tranquilli Leali and S. De Santis are gratefully acknowledged. The technical assistance of Mr. Dario Caporuscio, the photographic service of Mr. Luciano Falcinelli, the excellent secretarial activity of Ms. Silvana Casamento and the kind help of Mrs. Sharon Hobby in the revision of the English language are also gratefully acknowledged.
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ABBREVIATIONS GABA-t ␥-aminobutyric acid-transaminase MS multiple sclerosis BSA bovine serum albumin IL interleukin QAI quantitative analysis of images NAD nicotinamide adenine dinucleotide
INTRODUCTION Numerous experimental studies indicate that a link exists between the immune and neuroendocrine systems in mammals, including humans [1]. In both the primary and secondary lymphoid organs of rats, the norepinephrine amount is altered after an immune stimulating
From the 2nd Neurologic Clinic (D.C.), 3rd Surgical Clinic (V.D.), Department of Cardiovascular and Respiratory Sciences (C.C.), and Department of Pharmacology of Natural Molecules and General Physiology (M.A.), University “La Sapienza”, Rome, Italy. Address reprint requests to: Carlo Cavallotti, M.D., Department of Cardiovascular and Respiratory Sciences, University of Roma “La Sapienza”—Via A. Borelli, 50 00161 Rome, Italy; Tel: ⫹39 06 4958291; Fax: ⫹39 06 4957669; E-Mail: [email protected]. Received November 17, 1999; revised March 28, 2000; accepted March 30, 2000. Human Immunology 61, 697–704 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.
nerve fibers of the thymus. Treatment with interferon is also capable of increasing the protein content of the thymus. The biochemical data together with the histoenzymatic results provide evidence for a localization of GABA-t in the thymic gland. Moreover, ␥-aminobutyric acid can be considered as one of the linking molecules between the immune and nervous functions of the human thymus. Human Immunology 61, 697–704 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: ␥-aminobutyric acid-transaminase (GABA-t); immune response; thymus; interferon; quantitative analysis of images (QAI)
CU NADH IFNs IFN-
conventional units nicotinamide adenine dinucleotide hydrogenated interferons interferon-beta
therapy [2]. On the contrary, in immunodeficient mice some of the neurotransmitters of the thymus present numerous changes [3]. Weihe and coworkers [4], studying the molecular anatomy of the neuro-immune connection in a variety of mammals, demonstrated that numerous neuropeptides play a role as linking molecules between neuro-immune and neuro-endocrine functions of the thymic gland. ␥-Aminobutyric acid (GABA) can be considered as one of the linking molecules between the neural and neuroendocrine component of the human thymus [5]. GABA is widely recognized as a major inhibitory neurotransmitter in the vasculature of vertebrates [6]. GABA, its related enzymes, i.e., glutamate decarboxylase and ␥-aminobutyric acid-transaminase (GABA-t), as well as GABA 0198-8859/00/$–see front matter PII S0198-8859(00)00130-0
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receptors, have been demonstrated in a wide variety of peripheral tissues [7, 8]. Although the physiological relevance of GABA in most of the peripheral tissues remains to be clarified, there are convincing lines of evidence to suggest a role for GABA as an inhibitory neurotransmitter in the vertebrate peripheral nervous system [9]. In fact, GABA is present in the mouse thymus and undergoes a significant increase during the immune response, i.e., following challenge with a T-cell dependent antigen [10]. GABA-t activity in thymus of rats and mice has been demonstrated in our laboratories many years ago [11]. GABA-t is the enzyme that catabolizes GABA, a neurotransmitter that increases up to elevated levels in the thymus during an immune response [10]. In a recent article, we have demonstrated the occurrence of GABA-t also in nerve fibers of thymus from young and elderly men [12]. The purpose of this article is to study whether the increased levels of GABA found in the mouse thymus during the immune response [10] are confirmed by an increase of GABA-t in the human thymus after an immune-response. Moreover, the relationships between GABA-t and interferon-beta ( IFN-) in the human thymus in immunostimulated or in non-immunostimulated patients have been analyzed with special reference to the effects of immunomodulatory molecules interferons (IFNs) on neurotransmitter molecules (GABA). In fact, an increase in GABA-t content and the presence of IFN 1 may support the hypothesis that immunomodulatory molecules are able to exert some effects on neurotransmitters, thus providing reasonable evidence for a link between both systems. MATERIALS AND METHODS The experimental procedures performed in this study include: (1) patient’s immune stimulation, (2) thymic biopsies, (3) histochemical demonstration of GABA-t, (4) estimation of protein content, (5) biochemical dosage of GABA-t, (6) quantitative analysis of images, (7) statistical analysis of data. Each procedure is briefly explained. Patient’s Immune Stimulation In our city, many neurological hospitals are authorized as national data points for the study of multiple sclerosis (MS). In these hospitals, patients with MS are enrolled and treated by IFNs. -1a interferon (Avonex, SolvayDuphar, Olst, The Netherlands) belongs to a group of drugs that modulate the regulation of the human immune system. -1b interferon (Betaferon, Schering AG, Germany) also modifies the response of the human immune system. Recently, human recombinant  interferon (Serobif, Serono SPA, Italy) has been introduced
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into clinical practice as an immunomodulating systemic agent. All these mentioned interferons can be used in the treatment of patients affected by recurrent-remittent MS, whose clinical history is marked by at least two episodes of neurological involvement during the previous two years. In order to study the possible involvement of the thymus gland in MS, a number of patients with MS or affected by other neuroimmunological diseases (excluding myasthenia gravis), treated or untreated with interferon drugs, underwent (for diagnostic reasons) a surgical biopsy of the thymus. Small fragments of these thymic biopsies drawn from immunostimulated patients affected by MS or by other neurological diseases were thus available for our experiments. Our studies were approved by the Ethical Committees of the involved hospitals and patients also gave their informed written consent. In National Clinical Centers the following therapeutic procedures were used for administration of interferon drugs: 36 patients (age range 24 – 66 years) were randomly divided into three groups and treated by three different protocols in order to obtain a remission of their illness. Group 1: 14 patients enrolled; 6 of them treated for at least 6 months by one intramuscular injection of Avonex ( 1a interferon) weekly. Group 2: 12 patients enrolled; 5 of them treated for at least 6 months by three intradermal injections of Betaferon ( 1b interferon) 8.000.000 of International Units (IU) weekly. Group 3: 10 patients enrolled; 5 of them treated for at least 6 months (the drug is still completing the official registration course required by the Italian Health Authorities) by three intradermal or intramuscular injections of Serobif (Human recombinant) (-interferon) corresponding to 6.000.000 of IU weekly. Thymic Biopsies Three patients of the first group, two patients of the second group, and two patients of the third (all treated with interferons, n ⫽ 16) underwent surgical thymic biopsies. The remaining group of patients (n ⫽ 20) with MS was not treated by interferons because their symptoms did not conform to the protocols approved by Health Ministry for treatment by means of interferons. Seven patients affected by MS and untreated with interferons or affected by other neurological diseases also underwent diagnostic thymic biopsy. Samples from these patients were considered as untreated and served as the control group. Small fragments of all these biopsies were carried quickly under dry ice to our laboratories where they were submitted to the experimental procedures. Table 1 con-
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TABLE 1 Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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Relevant characteristics of 14 patients enrolled in our medical protocols
Treatment Avonex Avonex Avonex Betaferon Betaferon Serobif Serobif None None None None None None None
Age (yrs)
IgM (mg/dl ⫾ S.E.M.)
56 66 24 60 30 32 28 25 58 32 60 36 64 27
220 ⫾ 1.8 198 ⫾ 1.3 232 ⫾ 1.5 241 ⫾ 1.9 196 ⫾ 2.1 210 ⫾ 1.8 224 ⫾ 1.5 110 ⫾ 1.3 96 ⫾ 0.8 162 ⫾ 1.2 124 ⫾ 1.1 150 ⫾ 1.3 136 ⫾ 1.5 120 ⫾ 1.0
Diagnosis a
MS MSa MSa MSa MSa MSa MSa MS w.t.b MS w.t.b MS w.t.b MS w.t.b MS w.t.b MS w.t.b MS w.t.b
All patients underwent diagnostic thymic biopsy. Other 22 patients didn’t undergo thymic biopsy and so were excluded from the present experiments. IgM were tested many folds before the treatment and after 3, 4, 5, and 6 months from the beginning of therapy. The listed results are the mean values of four experiments (five for untreated patients) ⫾ SEM. The values before the treatment were excluded by treated patients, while were included for untreated patients. a MS ⫽ multiple sclerosis under treatment with -interferon owing to the clinical situation, as reported in Methods. b MS w.t. ⫽ multiple sclerosis without treatment.
tains all relevant characteristics of the patients enrolled in our medical protocols. Histochemical Demonstration of GABA-t Samples of human thymus coming from surgical biopsies in untreated or interferon-treated patients were rapidly placed in a dry-ice mixture for the histochemical demonstration of GABA-t activity. The Van Gelder method (1965) was used for histochemical detection of specific GABA-t [13], carrying out the slight modifications described in a previous paper of us to minimize enzyme diffusion [14]. In brief, each slice of thymus was stretched on preweighed slides, which were then newly weighed to estimate the weight of the sample. Samples were treated by histoenzymatic staining that involves the reduction of tetrazolium salts. Control sections were incubated without substrates or using, as inhibitors, 50 M of amino-oxalacetic acid or 50 M of Gabaculline or 200 M of acetylenic GABA or 100 M of ethanolamine sulfate, one at a time or simultaneously. Estimation of Protein Content In this group of experiments, the thymic biopsies were weighed and placed on dry ice, (specimens for GABA-t histochemical staining) or into an ice-cold homogenisation buffer (samples for estimating the protein content and the GABA-t activity).
Tissue protein concentrations were determined by the method described by Lowry et al. [15] using bovine serum albumin (BSA) as standard. Biochemical Dosage of GABA-t Biochemical dosage of GABA-t activity (EC 2.6.1.19 4-aminobutyrate 2-oxoglutarate aminotransferase) was carried out according to the method of Jung et al. [16] using 6 M GABA plus 5 M 2-oxoglutarate as substrate and determining the formation of NADH starting by NAD. Specific GABA-t activity was calculated by subtracting the blank values (measured in the presence of 50 M of amino-oxalacetic acid as inhibitor or in presence of other inhibitors or in absence of substrate) from the total activity. Other GABA-t inhibitors used were Gabaculline 50 M, acetylenic GABA 200 M or ethanolamine-o-sulfate 100 M. The biochemical values obtained using all these inhibitors (one at a time or simultaneously) can be considered as blank values and must be subtracted from the values of total activity. Quantitative Analysis of Images In order to evaluate the effects of immune-stimulation on GABA-t levels, a quantitative analysis of the intensity of the histochemical staining was performed on slides by means of a Quantimet Analyzer (Leica Microsystems Imaging Solutions, Cambridge, UK). The values of control sections, incubated without substrates, were considered as “zero.” Examinations were performed separately for each type of treatment on 7 treated patients and on 7 untreated patients, on at least fifteen slices for each patient, evaluating the standard error of the mean (⫾SEM). Final values must be submitted to statistical data analysis. The values reported represent the intensity of staining for each type of treatment and are expressed in conventional units (C.U.) ⫾SEM. Conventional units are arbitrary units that are furnished directly by the display of the Quantimet Analyzer [16]. Statistical Analysis of Data The statistical methods used throughout this study must be interpreted as an accurate description of the data rather than a statistical inference of such data. The preliminary studies of each value were performed with the of basic sample statistics. Mean values, maximum and minimum limits, standard deviation (SD), standard error of the mean (SEM) and correlation coefficients were carried out according to Serio [17]. The relationship between each pair of variables was studied using the respective correlation coefficients grouped in a
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FIGURE 1 GABA-transaminase activity in the human thymus of a young man (27 years old) untreated with interferons (Magnification 250⫻). Due to the thymic involution the picture shows (arrow) a small adipose tissue infiltration. The wall of many lymphatic vessels (L) shows a positive reaction. Numerous reticular cells (R) also show a positive reaction. Many structures resembling nerve fibers (n) also show a positive reaction. Medullary zone on the right side, cortical zone on the left side.
correlation matrix, thus enabling us to study the existence of a linear (values next ⫹1 or ⫺1) or non-linear (values next 0⫹) dependency. Finally, a correlative analysis of the morphological and biochemical data was performed by comparing the significant differences for each age with the corresponding values of the other homogeneous groups. Correlation coefficients denote a significant level less than 0.001 (p ⬍ 0.001) while the correlation coefficient is not significant when p ⬎ 0.05 (n.s.). This correlation coefficient was calculated according to Castino and Roletto [18]. RESULTS Our results are reported in Figures 1 through 4 and summarized in Tables 1 through 4. Figure 1 shows that in an untreated young man (27 years old) the GABA-t activity is localized in the wall of lymphatic vessels (L), in numerous reticular cells (R) and in many structures FIGURE 2 GABA-transaminase activity in the human thymus of an old man (60 years of age) untreated with interferons (Magnification 250⫻). We can observe an evident adipose tissue infiltration (arrows). The wall of two venules (V) and of two lymphatic vessels (L) shows a positive reaction. A Hassall’s corpuscle (H), considered as expression of thymic involution, shows a negative reaction, while many reticular cells (R) show only a few positive reactions. Many structures resembling nerve fibers (arrows) show a positive reaction. Medullary zone on the right side, cortical zone on the left side.
resembling nerve fibers (n). Thymic tissue shows a small adipose tissue infiltration (arrow). Figure 2 depicts an untreated old man (60 years of age) only two lymphatic vessels (L) and two veins (V) show a positive staining for the GABA-t activity. A corpuscle of Hassall (H) appears unstained. The reticular cells also show a positive reaction. Moreover, we can observe an evident adipose tissue infiltration (arrow). Figure 3, which must be compared with Figure 1, is a sample from a young patient (28 years of age) previously treated with interferon drugs. This photograph shows a strong positive reaction for GABA-t activity localized in reticular cells (R) and in the wall of an arteriola (A), of a venula (V) and of numerous lymphatic vessels (L). In Figure 4, we can observe strong positive enzymatic staining in reticular cells (R) in many structures resembling nerve fibers (n) and in the wall of an arteriola (A) and of a venula (V). The samples of thymus were taken
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FIGURE 3 GABA-transaminase activity in the human thymus of a young patient (28 years of age) treated with interferons (Magnification 250⫻). We can observe the signs of a thymic involution (arrow). The wall of an arteriola (A), of a venula (V), of numerous lymphatic vessels (L) and the reticular cells (R) show a positive reaction. Medullary zone on the right side, cortical zone on the left side.
from an old man (age 66 years) previously treated with interferon drugs. By comparing Figure 2 with Figure 4, it is evident that interferon treatment induced an increase of GABA-t levels. Table 1 deals with the relevant characteristics of 14 patients (which underwent diagnostic thymic biopsy) enrolled in our medical protocols. As can be seen treatment with INF- induces an immune response that can be verified by serum elevated IgM. Table 2 displays the results of protein content in homogenates of thymus in adult or old patients, either treated or untreated with interferon. As can be seen, therapeutic treatment with interferon drugs modifies the protein content of thymic homogenates both in adult and in older patients. In fact, in untreated adult patients protein levels expressed as mg/g fresh weight of thymus are 15.8 mg while in treated adult patients this level goes up to 19.6 mg with a highly positive treated versus untreated significance index (p ⬍ 0.001). Moreover, in FIGURE 4 GABA-transaminase activity in the human thymus of an old patient (66 years of age) treated with interferons (Magnification 250⫻). We can observe a strong positive reaction in reticular cells (R) while a Hassall’s corpuscle (H) shows a negative reaction. Structures resembling nerve fibers (n) show an evident positive reaction. Moreover, we can observe the wall of an arteriola (A) and of a venula (V) showing a positive reaction. Medullary zone on the right side, cortical zone on the left side.
untreated older patients, the thymus protein content is 10.4 mg/g tissue fresh weight. It goes up to 12.8 mg/g tissue fresh weight in thymus of patients treated with interferon drugs, with a positive index of significance despite the limited number of samples examined (3 treated and 3 untreated). Table 3 deals with the values of biochemical dosages of GABA-t activity in the thymus of adult and older patients treated and untreated with interferon. Results are expressed as nM /mg protein/h. In untreated men (n ⫽ 4, i.e., four subjects) the mean value was 45.6. It rose to 93.2 in patients treated with interferon. In untreated older men (n ⫽ 3, i.e., three subjects) the mean value was 22.4. It rose to 39.7 in patients treated with interferon. By comparing the significant differences between treated and untreated patients we can see that the significance index is highly positive p ⬍ 0.001. Table 4 shows the results of quantitative image anal-
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TABLE 2
Thymus Adult patients Age 24–36 n⫽4 Older patients Age 56–66 n⫽3
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Protein content in homogenates of thymus of adult and older patients treated and untreated with interferon Untreated (n ⫽ 7) 15.8 ⫾ 0.19 10.4 ⫾ 0.13
Treated with interferona (n ⫽ 7) 19.6 ⫾ 0.22b 12.8 ⫾ 0.18b
Results are expressed as mg/g of fresh weight tissue. Each value is the mean value ⫾ SD of independent determinations from n. patients, carried out in triplicate. n ⫽ number of subjects a Treatment of patients with interferon drugs was performed by one of the three procedures reported in Methods, alternatively. b p ⫽ ⬍ 0.001 treated versus untreated.
ysis of the GABA-t levels in biopsies of the thymus of adult and old patients in treated or untreated subjects. The mean value in four adult untreated patients was 28.9 C.U. It rose to 58.3 in four adult treated patients. In three untreated older patients the mean value of GABA-t levels was 13.6 C.U., rising to 26.4 C.U. in patients of the same age treated with interferon. These results show that after therapeutic treatment with interferon the level of GABA-t is increased. The biochemical data together with the histoenzymatical results provide evidence for a localization of GABA-t in the thymic gland. Moreover, the administration of interferon modifies the levels of GABA-t.
TABLE 3
Thymus Adult patients Age 24–36 n⫽4 Older patients Age 56–66 n⫽3
TABLE 4
Biochemical dosage of GABA-transaminase activity in thymus of adult and older patients treated and untreated with interferon Untreated (n ⫽ 7)
Treated with interferona (n ⫽ 7)
45.6 ⫾ 3.12
93.2 ⫾ 4.36b
22.4 ⫾ 1.63
39.7 ⫾ 2.84b
Results are expressed as nM/mg protein/h. Each value is the mean value ⫾ SD of independent determinations from n. patients, carried out in triplicate. n ⫽ number of subjects a Treatment of patients with interferon drugs was performed by one of the three procedures reported in Methods, alternatively. b p ⫽ ⬍ 0.001 treated versus untreated.
Thymus Adult patients Age 24–36 n⫽4 Older patients Age 56–66 n⫽3
Quantitative analysis of images of the GABA-transaminase levels in thymic biopsies of adult and older patients treated and untreated with interferon after histoenzymatical staining Untreated (n ⫽ 7)
Treated with interferona (n ⫽ 7)
28.9 ⫾ 2.6
58.3 ⫾ 3.1b
13.6 ⫾ 1.4
26.4 ⫾ 2.3b
Results are expressed in Conventional Units (C.U.) (see Methods) ⫾ SEM. The analyzer was calibrated considering “zero” the values of control sections incubated without substrate or in presence of specific inhibitors. For other precautions used see methods. p was calculated comparing the significant differences between treated and untreated patients. n ⫽ number of subjects a Treatment of patients with interferon drugs was performed by one of the three procedures reported in Methods, alternatively. b p ⫽ ⬍ 0.001 treated versus untreated.
DISCUSSION There are alternative histological methods for visualizing GABA-containing elements in mammalian tissues. It was generally assumed that radiolabeled GABA, taken up by a particular tissue, specifically labels the GABAcontaining cells that can be visualized by autoradiography . Recent findings have revealed, however, that the GABA-accumulating cells do not necessarily correspond to the GABA-containing and GABA synthesizing cells [19 –21]. Another approach is the immunocytochemical localization of glutamate decarboxylase, the marker enzyme of GABA containing structures. This procedure, however, also has some disadvantages. The topography of glutamate decarboxylase within a cell does not necessarily match the localization of the small, rapidly diffusing, GABA molecule: additionally, there are marked species and tissue differences in the immunochemical properties of the enzymes of different origin [22]. In addition, the distribution of the catabolic enzyme GABA,-t may not reflect the location of GABA within a particular tissue, but the enzyme-substrate reaction, generally used for visualization of GABA-t [13, 23], can be universally applied for different species and tissues. However, the direct GABA immunocytochemical method is capable of demonstrating relatively low tissue concentrations, is highly specific, and can be used both for light and electron-microscopic samples. Moreover, there is no problem with species and tissue specificity [24, 25]. Taking into account the advantages and disadvantages of the above procedures, the GABA-t histoenzymatic
GABA-t in Thymus and Interferons Treatment
method developed by Van Gelder [13] was adapted and used for studying the distribution of GABA-t in human thymus in normal conditions and after interferon administration. A low marginal GABA-t activity was found in association with thymic blood vessels. The enzymatic activity was higher in the thymic biopsies coming from treated subjects. In one of our recent publications [12], we demonstrated that GABA-t content appears to be increased with age. On the contrary, in this study a decrease of GABA-t in thymuses of old patients (if compared to young patients) is demonstrated. This apparent discrepancy may be explained because in these mentioned study, we used thymic fragments harvested from autopsies and considering the whole thymus, while, in the present study, specimens were drawn from surgical biopsies, obtaining very small fragments confined to the thymic microenvironment only. In fact, in this study we excluded other thymic structures (different from the parenchymal microenvironment) in which GABA-t is widely distributed. In contrast to IFN-␥, which is primary immunomodulator (with little anti-viral activity) INFs- are considered to be primarily anti-viral agents with some immunomodulatory activity. Nevertheless, the role of INF- in the modulation of the immune response in MS is well documented [26]. Moreover, antiviral activity of -interferon seems to exert a useful effect in the therapy of recurrent MS [27]. However, National Health Authorities recommend the use of -interferons in similar experimental protocols and so we are unable to modify these procedures. Treatment with interferon drugs induces an immune response. This response can be verified with dosages of serum elevated antibody titers (see Table 1) and by means of common laboratory tests performed during hospitalisation (protein immuno-electrophoresis a.s.o.). A significant rise in thymic GABA-t levels was found after treatment with interferon drugs. The potential biological significance of this increase can be considered. GABA-t activity catabolizes the GABA and was, therefore, strictly correlated with GABA metabolism. Since GABA is able to function as an inhibitory transmitter at the level of cholinergic receptors on the neuromuscular junction, it is possible that it might function in the same manner on the cholinergic receptors of the thymic epithelial cells that synthesize the thymosin. Moreover, large amounts of GABA have been found incorporated in many thymosin peptides that have been sequenced in their amino-acidic composition. In conclusion, our results demonstrate that patients treated with interferon, a potent promoter of immune responses, show high levels of GABA-t activity in thymic structures (vessels, nerve fibers and parenchyma) in
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comparison to untreated patients. Therefore, at present, the significance of the enhancing effect of INFs- on the presence of GABA-t remains very uncertain. Treatment with INF is also capable of increasing the protein content of the thymus. In fact, our data show that in vivo INF- treatment doubles the intrathymic amounts of GABA-t in humans. Is this effect thymusspecific? GABA-t is specifically enhanced in the wall of blood vessels in different vascular districts including cerebral vessels. Because we did not study other tissues, we are unable to exclude if the effects of interferon- are thymus-specific or not. What is the meaning of intrathymic expression of GABA-t? As previously mentioned, GABA is a neurotransmitter that plays a role in many central and peripheral nerve fibers as well as an amino-acid with several functions in protein metabolism. GABA-ergic systems in the thymus undergo changes after immunostimulating therapy. In our laboratory, experiments are currently under way to study variations of other neurochemical parameters (density of receptor binding sites, affinity of receptors to specific ligands, turnover of these substances). The related results may provide more valuable information about the meaning of intrathymic expression of GABA-t. Our biochemical data, together with histoenzymatic qualitative and quantitative results, provide direct evidence for a specific localization of GABA-t in the thymic gland. Moreover, reticular cells, epithelial cells and structures resembling nerve fibers of the thymus show a positive GABA-t staining. ACKNOWLEDGMENTS
The present study was supported by grants from MURST, CNR and University of Rome “La Sapienza” (COD-DIP 98.043.14). The authors are greatly indebted to Drs. Cristoforo Morocutti and Ricardo Miledi for their suggestions and criticisms. The Medlines, Internet and other informatic consulting services of Drs. M. Cameroni, F. M. Tranquilli Leali and S. De Santis are gratefully acknowledged. The technical assistance of Mr. Dario Caporuscio, the photographic service of Mr. Luciano Falcinelli, the excellent secretarial activity of Ms. Silvana Casamento and the kind help of Mrs. Sharon Hobby in the revision of the English language are also gratefully acknowledged.
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