Kg. Petersen et al., INSULIN AS A TARGET ANTIGEN IN AUTOIMMUNE DIABETES - A NATURAL REPERTOIRE AS THE SOURCE OF ANTIBODY-RESPONSE, Acta diabetologica, 31(2), 1994, pp. 66-72
A solid-phase immunoenzymatic technique with B1- or B29-biotinylated i
nsulin coupled to avidin coated wells was used to characterize serum a
nti-insulin antibodies and to locate insulin antibody-producing B lymp
hocytes in different organs of mice. Low natural serum anti-insulin Ig
M and IgG antibodies were found in ten different healthy inbred strain
s of mice. Prediabetic nonobese diabetic (NOD) mice had significantly
higher measurements than BALB/c mice (P < 0.05). Anti-insulin IgM anti
body-producing B lymphocytes were found in bone marrow and spleen of N
OD mice and healthy strains of mice, but not in peripheral lymph nodes
, thymus, blood or pancreas. B29-fixed insulin was more frequently rec
ognized than B1-fixed insulin. There was no relationship to the MHC or
to other immune markers. IgG insulin antibody-producing cells were no
t detected. IgG insulin antibody-producing cells appeared in the drain
ing lymph node and in the blood 10 days after immunization with insuli
n. IgM insulin-recognizing cells in the spleen were reduced in number
during the same period (P < 0.05-0.01 for BALB/c, DBA2, B10.D2 and NOD
), suggesting migration of these cells. This was tested by in vivo sta
ining of spleens with the red-fluorescent membrane linker PKH-26 on da
y 7 after immunization. Cells from immunized lymph nodes were FACS-sor
ted on day 10. Insulin antibody-producing B lymphocytes with red-fluor
escence were found, indicat ing a splenic origin. Examination of IgG s
ubclasses showed preferential production of complement-fixing IgG2b in
sera and lymph node cells of immunized NOD mice (P < 0.05 vs BALB/c).
We conclude that a natural repertoire of insulin recognition exists i
n bone marrow and spleen of mice. Hydrophobic epitopes around B1 (B29-
fixed insulin) are more frequently recognized than hydrophilic epitope
s around B29 (B1-fixed insulin), indicating a genetically fixed patter
n of autorecognition. Insulin-recognizing cells from the spleen functi
on as the source of insulin antibody response. Preferential occurrence
of complement-fixing IgG2b in NOD mice could contribute to autoimmune
-mediated beta-cell damage.