Casein Α S1 Is Expressed By Human Monocytes And Upregulates The Production Of GM-CSF Via P38 MAPK

Caseins are major constituents of mammalian milks that are thought to be exclusively expressed in mammary glands and to function primarily as a protein source, as well as to ameliorate intestinal calcium uptake. In addition, proinflammatory and immunomodulatory properties have been reported for bovine caseins. Our aim was to investigate whether human casein α s1 (CSN1S1) is expressed outside the mammary gland and possesses immunomodulatory functions in humans as well. For this purpose, CSN1S1 mRNA was detected in primary human monocytes and CD4+ and CD8+ T cells, but not in CD19 + B cells. CSN1S1 protein was traceable in supernatants of cultured primary human CD14+ monocytes by ELISA. Similarly, CSN1S1 mRNA and protein were detected in the human monocytic cell lines HL60, U937, and THP1 but not in Mono Mac 6 cells. Moreover, permeabilized human monocytes and HL60 cells could be stained by immunofluorescence, indicating intracellular expression. Recombinant human CSN1S1 was bound to the surface of Mono Mac 6 cells and upregulated the expression of GM-CSF mRNA in primary human monocytes and Mono Mac 6 cells in a time- and concentration-dependent manner. A similar increase in GM-CSF protein was found in the culture supernatants. CSN1S1-dependent upregulation of GM-CSF was specifically blocked by the addition of the p38 MAPK inhibitor ML3403. Our results indicated that human CSN1S1 may possess an immunomodulatory role beyond its nutritional function in milk. It is expressed in human monocytes and stimulates the expression of the proinflammatory cytokine GM-CSF. Copyright © 2010 by The American Association of Immunologists, Inc

Autoantibodies to aS1-Casein Are Induced by Breast-Feeding

Background: The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human aS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. Methods: 62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA. Results: IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SDELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless. Conclusion: We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breastfeeding, and sustained into adulthood

Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

BACKGROUND: The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human αS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. METHODS: 62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA. RESULTS: IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SD-ELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless. CONCLUSION: We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breast-feeding, and sustained into adulthood

Inhomogeneity of immune cell composition in the synovial sublining: linear mixed modelling indicates differences in distribution and spatial decline of CD68+ macrophages in osteoarthritis and rheumatoid arthritis

BACKGROUND: Inhomogeneity of immune cell distribution in the synovial sublining layer was analyzed in order to improve our mechanistic understanding of synovial inflammation and explore potential refinements for histological biomarkers in rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Synovial tissue of 20 patients (11 RA, 9 OA) was immunohistochemically stained for macrophages (CD68), synovial fibroblasts (CD55), T cells (CD3), plasma cells (CD38), endothelial cells (vWF) and mast cells (MCT). The synovial sublining layer was divided into predefined adjacent zones and fractions of the stained area (SA) were determined by digital image analysis for each cell marker. RESULTS: Distribution of CD68, CD55, CD38 and MCT staining of the sublining area was heterogeneous (Friedman ANOVA p < 0.05). The highest expression for all markers was observed in the upper layer close to the lining layer with a decrease in the middle and lower sublining. The SA of CD68, CD55 and CD38 was significantly higher in all layers of RA tissue compared to OA (p < 0.05), except the CD38 fraction of the lower sublining. Based on receiver operating characteristics analysis, CD68 SA of the total sublining resulted in the highest area under the curve (AUC 0.944, CI 95 % 0.844–1.0, p = 0.001) followed by CD68 in the upper and middle layer respectively (both AUC 0.933, CI 95 % 0.816–1.0, p = 0.001) in both RA and OA. Linear mixed modelling confirmed significant differences in the SA of sublining CD68 between OA and RA (p = 0.0042) with a higher concentration of CD68+ towards the lining layer and more rapid decline towards the periphery of the sublining in RA compared to OA (p = 0.0022). CONCLUSIONS: Immune cells are inhomogeneously distributed within the sublining layer. RA and OA tissue display differences in the number of CD68 macrophages and differences in CD68 decline within the synovial sublining

Serum reaction against human CSN1S1 in probands who were breast-fed for a short period of time.

<p>Eight probands who were breast fed for a short period of time (5 d–42 d), showed an increased antibody reaction against CSN1S1 in comparison to control persons, who were not breast-fed. (dotted line = cut-off value from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032716#pone-0032716-g004" target="_blank">Fig. 4</a>).</p

Antibody reaction against bovine αS1-casein.

<p>(A) Antibody reaction against <i>E. coli</i> UT5600(DE3) pSH3 (control, lane1), <i>E. coli</i> UT5600(DE3) displaying human CSN1S1 (lane 2) and E. coli UT5600(DE3) displaying bovine CSN1S1 (lane3) measured by SD-ELISA with a rabbit polyclonal anti- bovine CSN1S1 antibody. For detection a goat HRP conjugated anti rabbit antibody was used according to the conditions described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032716#pone-0032716-g003" target="_blank">Fig. 3</a>. (B) The antibody reaction against <i>E. coli</i> UT5600(DE3) pSH3 (control, black columns) and <i>E. coli</i> UT5600(DE3) displaying bovine CSN1S1 (white columns) were analyzed at different concentrations of the rabbit polyclonal anti-bovine CSN1S1 antiserum or in PBS with 3% FCS as negative control. (C) The SD ELISA against bovine CSN1S1 was repeated three times independently in triplicates at the highest antibody concentration applied (200 ng/ml) with <i>E. coli</i> UT5600(DE3)SH3 (control, lanes 1–3) and <i>E. coli</i> UT5600(DE3) displaying bovine CSN1S1 (lane4–6) in order to test the reproducibility.</p

Analyzing the IgM serum reaction against human CSN1S1 with respect to formula-fed and breast-fed test persons.

<p>61 of the 62 sera shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032716#pone-0032716-g003" target="_blank">Fig. 3</a> were divided into two consortia, a formula-fed and breast-fed, as it is shown for the IgG mediated response in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032716#pone-0032716-g004" target="_blank">Fig. 4</a>. The formula-fed consortium comprises the sera of 25 volunteers, whereas within the breast-fed consortium 36 sera were analyzed.</p

IgM reaction against human CSN1S1 in human sera.

<p>61 of the 62 sera shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032716#pone-0032716-g003" target="_blank">Fig. 3</a> with the IgG reaction on human CSN1S1 were analyzed for an IgM mediated serum response on the same antigen by SD-ELISA. Each serum was tested three times, the mean was calculated and shown as columns. SD of each mean is given as a line.</p

Surface accessibility of human CSN1S1 on <i>E. coli</i>.

<p>2.1) SDS-PAGE of outer membrane protein preparations from <i>E. coli</i> UT5600(DE3) pKP010 which were incubated without trypsin (lane 1) and cells which were treated with trypsin (lane 2). OmpF/C and OmpA are natural outer membrane proteins of <i>E. coli</i>, and can be used as reporters for successful outer membrane protein preparations. 2.2) Flow cytometer analysis of <i>E. coli</i> cells displaying αS1-casein. (A) <i>E. coli</i> UT5600(DE3) pKP010 were incubated with a polyclonal rabbit antiserum against CSN1S1 casein and subsequently with a secondary antibody conjugated with FITC. (B) The mean fluorescence of <i>E. coli</i> UT5600(DE3) pKP010 was 10,071. The value of the fluorescence for <i>E. coli</i> UT5600(DE3) cells without a plasmid, used as a negative control, was 464. 2.3) Fluorescence microscopy of both cell types. The cells were treated identically as described for flow cytometer analysis (2.2). (A) <i>E. coli</i> UT5600(DE3) pKP010 ; (B) <i>E. coli</i> UT5600(DE3) as negative control; (C) <i>E. coli</i> UT5600(DE3) as negative control, transmission light control.</p