autoimmune stats: DETECTION OF NOVEL ANTI NEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA) IN RAT LIVER

This text is based on the contents of a poster presented at the 25th National Congress of the Spanish Society of Immunology held in Torremolinos in the year 1999. An abstract in Spanish was published at the same event by J Rosique Román and R Alvarez López.

SUMMARY

Introduction: ANCA have recently been shown to be essential for the diagnosis and follow-up of a group of chronic inflammatory disorders. The most useful known targets are proteinase 3 (PR3) for Wegener’s disease or myeloperoxidase (MPO) for microscopic panarteritis nodosa (MPAN). There are a number of other ANCA specificities for which lighter associations have been reported with such autoimmune disorders as lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease or primary sclerosing cholangitis. In clinical practice serum samples are routinely screened for their presence by indirect immunofluorescence (IIF) techniques using ethanol-fixed human neutrophil smears. Little is known about the etiology or build-up of the ANCA immune response.

Methods: During the last two and a half years a total of 15000 sera were submitted to our laboratory for the study of non-organ specific autoantibodies. IIF testing was performed through standard procedures on rat stomach, kidney cortex and liver.

Results: 369 patients showed a cytoplasmic staining of polymorphonuclear cells scattered within rat liver sinusoids or less frequently within kidney glomeruli or just inside any vessel. In 158 of these patients a characteristic gastric mucosal granular cytoplasmic staining was also apparent. Other associated findings were: positive ANCA, positive biliary duct granular staining in rat liver, zona pellucida staining in monkey ovary tissue.

Conclusion: there are more ANCA than those currently detectable in routine testing procedures. They be significant for the pathogenesis of ANCA-associated inflammatory disorders or take part in antigen extension pathways. Shared antigenicity between neutrophils and biliary ducts may be relevant for the understanding of primary sclerosing cholangitis-associated ANCA.

INTRODUCTION

Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies that can be detected in human serum samples by indirect immunofluorescence (IIF) testing on human neutrophils. By using ethanol-fixed neutrophils two main patterns have been described, “c” or cytoplasmic, and “p”, perinuclear, which must be differentiated from other true antinuclear antibodies. A number of protein targets have been already reported, such as proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin, elastase, cathepsin G, bactericidal/permeability-increasing protein (BPI), lysozyme and azurocidin. PR3 antibodies are routinely ordered in clinical practice because of their association with Wegener’s disease, whereas MPO antibodies are useful for the management of microscopic panarteritis nodosa (MPAN) and rapidly progressive glomerulonephritis (RPGN). They are also commonly found in Goodpasture’s glomerulonephritis. The other specificities have less diagnostic value and have been unconsistently associated with lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease (Crohn or ulcerative colitis), primary sclerosing cholangitis or other vasculitides (Churg-Strauss, Takayasu, PAN). A pathogenic mechanism has been suggested by which the neutrophil plays a prominent role by undergoing activation and degranulation and causing damage to “innocent” by-standing tissues. There are also reports that support the possibility of endothelial expression of these autoantigens.

In our setting ANCA are routinely screened through IIF testing on ethanol-fixed neutrophils. Positive samples may undergo further characterization and quantitation with the use of a battery of enzyme immunoassays. It is recommended that the presence of non-organ specific nuclear and cytoplasmic autoantibodies be excluded by other IIF tests, thus helping to differentiate anti-cytoskeleton or anti-ribosomal autoantibodies from c-ANCA patterns, or antinuclear antibodies from p-ANCA. In case there is doubt about the purity of the antigen in any enzyme immunoassay, Western blot or immunoprecipitation studies may be necessary. For clinical routine it is acceptable that an experienced reader be appropriately trained for recognizing the microscopy patterns of the most clinically important specificities but cautious interpretation of results by the clinician is also advisable. Antibody-captured antigen assays may prove to play an essential role for the quantitation of antibodies recognizing conformation-dependent epitopes.

By IIF testing on rat stomach, kidney cortex and liver it is very common that a group of heterophile antibodies are found that recognize the brush border of rat proximal renal tubules. Some of the patients display specially brisk responses that extend to rat liver sinusoids and rat stomach endomysium and are thus reported as Rs reticulin antibody positive.

Zona pellucida autoantibodies have been described and proposed as a mechanism of female infertility. A group of bullous skin diseases have been shown to associate with the presence of autoantibodies directed to squamous epithelial intercellular substances (ICS).

MATERIAL AND METHODS

Patients:

We have computed the last 15000 sera that were sent to our laboratory for non-organ specific autoantibodies or RA testing (approximately after June 1996).

Antibodies assessments:

Non-organ specific antibodies: IIF procedure was performed on rat stomach, renal cortex and liver tissue slides (Biosystems, Barcelona). The initial dilution of sera for screening was 1/40. Bound antibodies were then stained with FITC-conjugated anti-human G,A,M, kappa, lambda antibody.

Other organ specific antibodies were similarly determined by IIF on monkey oesophagus, pancreas or ovary slides (Biosystems, Barcelona), or ethanol-fixed neutrophils (Inova). Sometimes a monkey preabsorbed FITC conjugate was used to minimize nonspecific background staining.

RESULTS

369 patients showed a cytoplasmic staining of polymorphonuclear cells that could be spotted within rat liver sinusoids or less frequently in kidney glomeruli or just inside any vessel. A halo was commonly observed around these cells as if the antigen of interest was diffusing away from its original location.

Of those patients, 40% also showed a granular cytoplasmic pattern of gastric (principal) mucosal cells, sometimes stronger at the luminal side. Similarly strong staining of clumps of secretory material within the glands may also be observed at this site. Most high-titer sera also display a granular staining of biliary ducts in liver portal areas.

This pattern could be seen – with difficulty, because of the high background staining – very commonly in sera with high titer heterophile “brush border” and reticulin Rs antibodies.

The finding of antinuclear, smooth muscle, mitochondrial or reticulin antibodies in the same serum was possible but deemed coincidental.

The strongest sera were also tested in several monkey tissues. Common findings in monkey oesophagus were positive staining for squamous epithelium SIC, or capillary vessel “dots” between muscular bundles. In monkey pancreas we commonly detected enhanced staining of exocrine acinar secretory granules. In monkey ovary tissue the most prominent staining usually was in the vascular spaces aroung tertiary or Graafian follicles or within the zona pellucida around both actively developing or atresic oocytes.

Routine ANCA testing in ethanol- or formalin-fixed neutrophils was typically negative but for a few cases. However, between the neutrophils in the same slides some unidentified amorphous “ghost” round bodies might show a strong positive staining. One of the p-ANCA positive sera was finally diagnosed as having MPO antibodies.

CONCLUSION

A new pattern of antibodies can be defined by IIF testing on rat liver, stomach and kidney which consists of polymorphonuclear cells with a halo around them, gastric mucosal granular cytoplamic and biliary duct staining. These antibodies may also be considered a new type of ANCA.

This immune response might prove to be an extension of one of the commonest “natural” human antibody responses, i.e. that of “rat heterophile” or “brush border” antibodies. It is worth investigating whether this group of antibodies can initiate any type of immune recognition in squamous epithelial SIC, zona pellucida, or endothelia that ultimately lead to more severe or clinically significant chronic inflammatory diseases.

There probably exists an antigen shared between rat neutrophils, gastric mucosa and biliary ducts. If the corresponding antigen can be found in human cells it should be investigated as a target of the primary sclerosing cholangitis-associated immune response.