A role of IgM antibodies in monosodium urate crystal formation and associated adjuvanticity

Abstract

Uric acid is released from injured cells and can act as an adjuvant signal to the immune system. Uric acid crystals invoke strong inflammatory responses in tissues. Although their biological effects are evident and the associated signaling mechanisms are becoming clear, it remains unexplained as to why uric acid precipitates rapidly in vivo, in sharp contrast to the minimal crystallization in vitro. We report in this study that a group of IgM Abs is able to bind to these crystals, which is interesting in light that B cell-deficient mice do not sense the proinflammatory adjuvant effect of uric acid. The titers of these Abs increase upon immunization with uric acid crystals. We have produced large quantities of such mAbs. The purified IgM Abs can significantly facilitate uric acid precipitation to form the inflammatory crystals in vitro. Infusion of these Abs into B cell-deficient mice significantly increases the basal level of inflammation in these recipients and restores the host's ability to sense uric acid's adjuvanticity. Therefore, we have identified a factor in determining uric acid precipitation and possibly its ability to function as an endogenous adjuvant. This finding suggests a new mechanism of the pathogenesis of gouty arthritis and uric acid-induced immune activation.

Figure 1. B cell deficient mice do not sense uric acid as an adjuvant

WT or muMT mice were immunized s.c. in the hinder flanks with 5 ug of OVA/latex beads mixed with PBS, 100 ug of MSU crystals or uric acid solution (100 ul total volume). 7 days later, the mice were sacrificed and 50 million splenocytes were prepared and stimulated with the 10⁻¹⁰ SIINFEKL peptide. 5 days later, the resulting T cells were counted and the target EL4 (H-2B) cells were labelled with ⁵¹Cr and pulsed with the same peptide. A 5 hr CTL assay was performed at the E:T ratio indicated, and as described in the Methods. The two lines in each panel represent two independent mice. The data are representative of 3 independent repeats. Most of error bars (1 standard deviation) were covered by the symbols.

Figure 2. The cloning and analysis of MSU crystal binding antibodies

A. Unimmunized B6 or immunized Balb/c or B6 serum (25 ul) was used to stain 500 ug of MSU crystals, followed by a 2nd FITC conjugated antibody recognizing mouse IgG/IgM H+L chains. The FACS analysis is similar to a typical cell-based assay except the crystals are smaller on the forward scatter. The lightly shaded area in this and following assays is 2nd antibody only control. B. Identical to A except xanthine crystals were used in place of MSU. C. Supernatant from a representative high binding UBA (UBA 11, 100 ul) was used to stain MSU crystals as in A. Imm: MSU immune serum. D. Schematic representation of the frequencies of various immunoglobulin subtypes within MSU binding mAbs.

Figure 3. UBAs recognize MSU crystals

A. Similarly immunized muMT serum was compared with an UBA (UBA E6) or a 20 fold dilution of UBA (UBA/D) E6. B. Human (W6/32) or mouse (Y3) MHC class I specific control antibodies do not stain MSU crystals.

Figure 4. UBAs precipitate soluble uric acid

1 mg/ml uric acid solution in pH 8.0 PBS was incubated with the indicated concentrations of UBA 11 and OVA for 6 hours in 24 well tissue culture plates. The MSU crystals were absorbed at the bottom of the wells (via antibody binding to the plate). The solution was aspired and the wells were washed with PBS. The deposited MSU crystals were resuspended in 0.01 N NaOH and prepared as described in the methods, and the UV absorption value at 292 nm was determined by a spectrophotometer. The amount of crystal deposition in microgram was converted from a standard UV absorption curve with known uric acid quantities.

Figure 5. Intact IgM structure is required for MSU crystal precipitation

A. UBA E6 digestion fragments (equal starting amounts) were used to stain MSU crystals. Assays with fragment specific 2nd antibodies produced the same results (data not shown). B. Similar to Figure 4, except that IgM fragments (equal starting amounts) or additional controls (OVA or MOPC control IgM) were used to analyze MSU precipitation. Filled empty bars were two independent replicates. C. Cultured bone marrow GMCSF/IL-4 DCs were stimulated with MSU and with the addition of indicated mAb supernatants for 6 hours. CD11c positive cells were analyzed for their CD86 expression by FACS.

Figure 6. UBAs increase the inflammatory indicators in muMT mice

A. B6 mice were injected i.v. with soluble uric acid (1 mg per day) and indicated antibodies (500 ug per day). UA: uric acid; E6C7: UBA; cAB: control IgM. Serum uric acid levels were analyzed by HPLC as described in Methods. B. The lung tissue from test C57BL/6 mice were homogenized and analyzed as described in the methods section. The myeloperoxidase values were generated by multiplying the UV reading by a factor of 0.2528 per a standard protocol. C. The serum C3 levels in test muMT mice after 3 days of uric acid/antibody infusion were determined using an ELISA kit (see Methods). D. Blood CD11b+ cells (neutrophils) were gated and analyzed for their CD62L expression.

Figure 7. UBAs enable muMT mice to sense uric acid as an endogenous adjuvant

The muMT mice were pre-injected i.v. with 2× of 500 ug purified UBA E6, a control IgM or PBS prior to the immunization s.c. in the hinder flanks with 5 ug of OVA/latex beads admixed with 100 ug of MSU crystals. An assay identical to that in Figure 1 was performed. Control EL4 targets without the peptide pulsing produced no significant reading. The two lines in each panel represent two independent mice. The data are representative of 3 independent experiments.