Insulin autoantibodies with high affinity to the bovine milk protein alpha casein

Abstract

Insulin autoantibodies (IAA) can appear in children within months of introducing solid foods to the diet and before clinical type 1 diabetes. The aim of this study was to determine whether infant dietary antigens could be immunizing agents of IAA. To this end, IAA binding to [(125) I]insulin was competed with food preparations and extracts of foods encountered in the infant diet (milk formulas, bovine milk, wheat flour, fowl meal). Bovine milk powder extracts inhibited IAA-positive samples from six of 53 children (age 0·3-14·0 years) participating in German prospective cohorts. Inhibition in these sera ranged from 23 to 100%. Competition was abolished when hydrolyzed milk powder was used. Competition with protein components of bovine milk found that two of the six milk-reactive sera were inhibited strongly by alpha- and beta-casein; none were inhibited by the milk proteins bovine serum albumin or lactoglobulins. The two casein-reactive sera had high affinity to alpha-casein (1·7×10(9) ; 3·1×10(9) l/mol), and lesser affinity to beta-casein (4·0×10(8) ; 7·0×10(7) l/mol) and insulin (2·6×10(8) ; 1·6×10(8) l/mol). No children with milk-reactive IAA developed autoantibodies to other islet autoantigens or diabetes (median follow-up 9·8 years). These results suggest that autoimmunity to insulin can occur infrequently via cross-reactivity to food proteins, but this form of IAA immunization does not appear to be associated with progression to diabetes.

Trial registration: ClinicalTrials.gov NCT01115621.

Fig. 1

Competition of insulin autoantibody (IAA) binding to human [¹²⁵I]insulin by addition of different food protein extractions (wheat flour, fowl meal, whole bovine milk, regular non-hydrolyzed (REG) and hydrolyzed (HA) infant milk formula, 100 µg/ml) in comparison to human insulin (2·2 × 10⁻⁵ mol/l). Results are shown as binding (cpm) without competition and binding in the presence of the extracts. Results from seven sera selected because of their heterogeneous binding to insulin, as reported previously [22,24], are shown together with sera from two patients with type 1 diabetes. Two of the seven children were inhibited strongly (100%, 93%) and three were inhibited weakly by whole bovine milk powder and milk formulas (marked by arrows).

Fig. 2

Individual insulin autoantibody (IAA) binding characteristics of one selected sample. Competition of binding to human [¹²⁵I]insulin by (a) protein extractions from food components [wheat flour, whole bovine milk and four representative infant formulas – regular non-hydrolyzed formula (REG), hypo-allergic hydrolyzed milk formula (HA), infant formula closely adapted to human breast milk (PRE), milk-free infant formula (SOM)] and (b) by milk protein fractions [alpha-casein, beta-casein, lactoglobulin and bovine serum albumin BSA)] are shown. IAA are expressed as bound radioactivity in counts per minute (cpm).

Fig. 3

Competition of binding to human [¹²⁵I]insulin by alpha- (dotted line) and beta-casein (grey line) in comparison to human insulin (black line) from the six children with insulin autoantibodies (IAA) that bind to whole bovine milk; in (a) subject 2, (b) subject 3, (c) subject 5, (d) subject 6, (e) subject 7 and (f) subject 8. The affinities to the competing protein are indicated by the numbers within the figures (l/mol). IAA in (a) and (d) bind better to alpha-casein than to human insulin, and in (b), (c), (e) and (f) bind to human insulin only. IAA are expressed as bound radioactivity in counts per minute (cpm).

Fig. 4

Detection of CD4⁺ T cell antigen-specific responses. (a) Representative fluorescence activated cell sorter (FACS) plots of carboxyfluorescein succinimidyl ester (CFSE) dilution obtained in subject 2. T cells were labelled with CFSE and incubated with alpha-casein or no antigen (medium). In response to alpha-casein, most of the proliferating CD4⁺CFSE^dim T cells (Q1; upper panel) also up-regulated the activation marker CD25 and memory marker CD45RO (Q2; lower panel) underlining the specificity of the T cell response. (b) The percentage of CD4 T cells which were CFSE^dim and CD25⁺CD45RO⁺ after 7-day culture with alpha-casein (5 µg/ml), beta-casein (5 µg/ml), bovine serum albumin (BSA) (5 µg/ml), proinsulin (10 µg/ml), tetanus toxoid (TT, 1 µl/ml) or medium only in subjects 2 and 5 and patients T1D_·1 and T1D_·2 from Fig. 1.