Clin. exp. Immunol. (1992) 90, 85-87
Absence of circulating interferon in patients with inflammatory bowel disease. Suggestion against an autoimmune etiology M. R. CAPOBIANCHI, S. FAIS*, M. C. DI PAOLO*, D. AGOSTINI*, P. PAOLUZI*, F. PALLONEt & F. DIANZANI Istituto di Virologia and *Istituto di Clinica Medica 2, Universitai 'La Sapienza', Roma, and tDipartimento di Medicina Sperimentale, Universitb di R. Calabria, Catanzaro, Italy
(Acceptedfor publication 2 July 1992)
SUMMARY Whether inflammatory bowel diseases (IBD) can be classified as autoimmune disorders is not established. Since circulating acid-labile interferon-alpha (IFN-a) is believed to reflect autoimmune reactions, we tested sera from two groups of IBD patients for the presence of circulating IFN. No detectable IFN was found in 51 serum samples of IBD patients. Furthermore, in no serum sample of IBD patients were neutralizing anti-IFN antibodies found. In contrast, acid-labile IFN-a was present in sera from 21/52 HIV-infected and from 6/14 systemic lupus erythematosus patients. These observations provide evidence that IBD differs from systemic autoimmune disorders, at least for the presence of circulating IFN. Keywords inflammatory bowel disease neutralizing antibodies autoimmunity patients with CD and in 15 with UC while 15 CD and 9 UC patients were on steroids at the time of the study. Eight of the 51 IBD patients had active extraintestinal manifestations. Sera from 52 patients infected with HIV (CDC II and III) and from 14 SLE patients were tested for comparison. Sera from whole venous blood were stored at - 80'C and tested within 1 month.
INTRODUCTION Interferon (IFN) plays an important role in primary surveillance against viral infections and influences several immune mechanisms [1]. Circulating IFN has been reported in several autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, systemic vasculitis and pemphigus [2]. Circulating IFN has also been found in HIVinfected patients, correlating with disease progression [3]. This IFN is of the alpha type, but shows abnormal sensitivity to acid treatment, and has therefore been termed acid-labile IFN-a [4]. Although not included in the list of criteria suggestive of autoimmune disorders, circulating acid-labile IFN-a is believed to reflect ongoing autoimmune reactions [5]. The etiology of inflammatory bowel diseases (IBD) is unknown. An autoimmune basis has been postulated, based on the presence of several indicators of autoimmunity [6]. However, IBD do not fulfil all criteria for classification as autoimmune disorders [6] and analogies between IBD and the classical autoimmune diseases are of interest. In this study serum samples of Crohn's disease (CD) and ulcerative colitis (UC) patients were tested for the presence of circulating IFN and of antibodies to it.
IFN assays IFN titration was performed on human WISH cells by inhibition of Sindbis virus haemagglutinin yield, after a single growth cycle [7] and standardized using NIH Ga 023-902-530 human leucocyte IFN-a as reference. The anti-viral activity was characterized by incubation with the following specific antisera: polyclonal sheep anti-IFN-a (Schering Co.); polyclonal rabbit anti-IFN-y (Dr J. Wietzerbin, Paris, France); monoclonal antiIFN-y (Hoffman-LaRoche). To test IFN stability at pH 2, 80 MI aliquots of IFN were adjusted to pH 2 by addition of 80 il 0 1 N HCl in RPMI, incubated for 24 h at 4°C, and returned to pH 7 with 40 pl RPMI containing 02 N NaOH. Reference human IFN-a and -# were completely stable during this procedure, whereas human IFN-y was strongly inactivated [8].
Neutralizing IFN antibodies
Neutralizing antibody titres were determined by a neutralization test against 5 U of either rIFN-cx2B, or rIFN-y [9]. After inactivation at 56°C for 30 min 60 pil of two-fold dilutions of
PATIENTS AND METHODS
Patients
sample or control sera were incubated at 37°C with 60 p1 of each type of IFN. After 1 h, 100 p1 of each mixture were added to duplicate monolayers of human WISH cells in 96-well microtitre plates. After overnight incubation, cells were challenged
patints ith wee C teted.The iseae wa actve i 14
Correspondence: Maria R. Capobianchi, PhD, The Institute of Virology, Viale di Porta Tiburtina, 28, 00185 Rome, Italy.
85
86
M. R. Capobianchi et al.
Table 1. Biological characterization of anti-viral activity found in sera from HIV-infected and systemic lupus erythematosus (SLE) patients
infected individuals and of SLE patients, chosen as a positive
Residual IFN titre (U/ml) after Serum sample no.
We failed to show either circulating IFN or anti-IFN neutralizing antibodies in serum samples from such patients. In contrast, we were able to detect circulating IFN in about 40% of HIV-
No treatment
Treatment at pH 2
Anti-IFN-a
Anti-IFN-y
1* 2* 3*
10 64 64
Absence of circulating interferon in patients with inflammatory bowel disease. Suggestion against an autoimmune etiology M. R. CAPOBIANCHI, S. FAIS*, M. C. DI PAOLO*, D. AGOSTINI*, P. PAOLUZI*, F. PALLONEt & F. DIANZANI Istituto di Virologia and *Istituto di Clinica Medica 2, Universitai 'La Sapienza', Roma, and tDipartimento di Medicina Sperimentale, Universitb di R. Calabria, Catanzaro, Italy
(Acceptedfor publication 2 July 1992)
SUMMARY Whether inflammatory bowel diseases (IBD) can be classified as autoimmune disorders is not established. Since circulating acid-labile interferon-alpha (IFN-a) is believed to reflect autoimmune reactions, we tested sera from two groups of IBD patients for the presence of circulating IFN. No detectable IFN was found in 51 serum samples of IBD patients. Furthermore, in no serum sample of IBD patients were neutralizing anti-IFN antibodies found. In contrast, acid-labile IFN-a was present in sera from 21/52 HIV-infected and from 6/14 systemic lupus erythematosus patients. These observations provide evidence that IBD differs from systemic autoimmune disorders, at least for the presence of circulating IFN. Keywords inflammatory bowel disease neutralizing antibodies autoimmunity patients with CD and in 15 with UC while 15 CD and 9 UC patients were on steroids at the time of the study. Eight of the 51 IBD patients had active extraintestinal manifestations. Sera from 52 patients infected with HIV (CDC II and III) and from 14 SLE patients were tested for comparison. Sera from whole venous blood were stored at - 80'C and tested within 1 month.
INTRODUCTION Interferon (IFN) plays an important role in primary surveillance against viral infections and influences several immune mechanisms [1]. Circulating IFN has been reported in several autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, systemic vasculitis and pemphigus [2]. Circulating IFN has also been found in HIVinfected patients, correlating with disease progression [3]. This IFN is of the alpha type, but shows abnormal sensitivity to acid treatment, and has therefore been termed acid-labile IFN-a [4]. Although not included in the list of criteria suggestive of autoimmune disorders, circulating acid-labile IFN-a is believed to reflect ongoing autoimmune reactions [5]. The etiology of inflammatory bowel diseases (IBD) is unknown. An autoimmune basis has been postulated, based on the presence of several indicators of autoimmunity [6]. However, IBD do not fulfil all criteria for classification as autoimmune disorders [6] and analogies between IBD and the classical autoimmune diseases are of interest. In this study serum samples of Crohn's disease (CD) and ulcerative colitis (UC) patients were tested for the presence of circulating IFN and of antibodies to it.
IFN assays IFN titration was performed on human WISH cells by inhibition of Sindbis virus haemagglutinin yield, after a single growth cycle [7] and standardized using NIH Ga 023-902-530 human leucocyte IFN-a as reference. The anti-viral activity was characterized by incubation with the following specific antisera: polyclonal sheep anti-IFN-a (Schering Co.); polyclonal rabbit anti-IFN-y (Dr J. Wietzerbin, Paris, France); monoclonal antiIFN-y (Hoffman-LaRoche). To test IFN stability at pH 2, 80 MI aliquots of IFN were adjusted to pH 2 by addition of 80 il 0 1 N HCl in RPMI, incubated for 24 h at 4°C, and returned to pH 7 with 40 pl RPMI containing 02 N NaOH. Reference human IFN-a and -# were completely stable during this procedure, whereas human IFN-y was strongly inactivated [8].
Neutralizing IFN antibodies
Neutralizing antibody titres were determined by a neutralization test against 5 U of either rIFN-cx2B, or rIFN-y [9]. After inactivation at 56°C for 30 min 60 pil of two-fold dilutions of
PATIENTS AND METHODS
Patients
sample or control sera were incubated at 37°C with 60 p1 of each type of IFN. After 1 h, 100 p1 of each mixture were added to duplicate monolayers of human WISH cells in 96-well microtitre plates. After overnight incubation, cells were challenged
patints ith wee C teted.The iseae wa actve i 14
Correspondence: Maria R. Capobianchi, PhD, The Institute of Virology, Viale di Porta Tiburtina, 28, 00185 Rome, Italy.
85
86
M. R. Capobianchi et al.
Table 1. Biological characterization of anti-viral activity found in sera from HIV-infected and systemic lupus erythematosus (SLE) patients
infected individuals and of SLE patients, chosen as a positive
Residual IFN titre (U/ml) after Serum sample no.
We failed to show either circulating IFN or anti-IFN neutralizing antibodies in serum samples from such patients. In contrast, we were able to detect circulating IFN in about 40% of HIV-
No treatment
Treatment at pH 2
Anti-IFN-a
Anti-IFN-y
1* 2* 3*
10 64 64
