Volume 57, Issue 5, Pages 1936-1948 (May 2000) Identification of a novel Fcα receptor expressed by human mesangial cells  Jonathan Barratt, Morag R. Greer, Izabella Z.A. Pawluczyk, Alice C. Allen, Elaine M. Bailey, Katharine S. Buck, John Feehally  Kidney International  Volume 57, Issue 5, Pages 1936-1948 (May 2000) DOI: 10.1046/j.1523-1755.2000.00043.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 IgA binding to cultured human mesangial cells (HMCs). Cells were detached and washed in PBS/BSA/sodium azide (P/B/A) before being incubated for one hour at 4°C with a range of IgA-FITC concentrations. Mesangial IgA binding is presented as molecules of equivalent soluble fluorescein (MESF) ± SEM and corresponds to five separate experiments each performed in triplicate on a single HMC cell line. IgA binding was concentration dependent and saturation was achieved at 50 μg/mL of IgA-FITC. Similar results were obtained with each of the other four HMC lines. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 Determination of the specificity of IgA binding to cultured HMCs. Mesangial cells were detached, washed, and incubated for 45 minutes at 4°C with an excess of unlabelled inhibitor protein before assessment of binding with IgA-FITC (50 μg/mL). Data are expressed as the percentage inhibition produced by each inhibitor protein compared with binding of IgA-FITC in the absence of inhibitor. Results shown are means ± SEM of four different experiments performed on a single HMC line. No inhibition was seen with BSA, IgG, IgM, IgA Fab fragment, or ASF. Only intact IgA and Fcα fragment were able to inhibit the binding of IgA to HMC, and this was consistently seen with all five primary HMC cultures. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Binding of different IgA isoforms to HMCs. Equal numbers of cells were incubated for 60 minutes at 4°C with 1 mg/mL of each IgA isoform, except mIgA1 (2.0 mg/mL) and binding detected with a FITC-F(ab′)2 antihuman IgAα chain antibody. Data represent the MCF ± SEM of four different experiments using the same HMC line. HMCs were capable of binding all isoforms of IgA equally; however, pIgA bound with a much greater affinity than any other IgA isoform. These findings were consistent across the five different primary mesangial cell cultures. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 My43 mediated inhibition of IgA binding to HMC (A) and PMA-activated U937 cells (B). Cells were washed at 4°C and IgA (50 μg/mL) binding assessed after a 45-minute incubation with either My43 or P/B/A. Viable cells were analyzed for IgA binding by flow cytometry, as described in theMethods section. In both panels, the shaded area represents the autofluorescence of U937 or HMCs, and the overlaid histograms correspond to the IgA binding obtained following incubation with either My43 or P/B/A. My43 was unable to inhibit IgA binding to HMCs; in contrast, there was a marked reduction in binding of IgA to PMA-activated U937 cells following preincubation with My43. Identical results were obtained when HMCs from each of the other primary mesangial cultures were used. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Detection of CD89 protein synthesis by HMCs. (A) Mesangial cells were stained for cell surface expression of CD89 (▪), and intracellular flow cytometry was performed to identify cytoplasmic CD89 (▪). Cell surface expression of HLA class I and intracellular staining with an antinucleus antibody (ANA) were used as positive controls. The results shown are MCF ± SEM of five different experiments performed on a single HMC line. At no time could we demonstrate the presence of CD89 expression by HMCs, despite clear evidence of cell surface and cytoplasmic staining for control proteins. Identical results were obtained with all five primary HMC cultures. (B) For comparison, these experiments were repeated using PMA-activated U937 cells. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 6 Attempted induction of latent CD89 synthesis by HMCs. Mesangial cells were rendered quiescent for 72 hours prior to being incubated for 48 hours with increasing concentrations of PMA, LPS, IFN-γ, TNF-α, or IL-6. IgA binding (A) and CD89 (combined cell surface and cytoplasmic) expression (B) were then measured by flow cytometry. To demonstrate adequate mesangial activation, staining for HLA classes I and II was also performed. In this experiment, HMCs were incubated with IFN-γ (5 ng/mL) and IgA binding (▾), and induction of CD89 or HLA staining (▪ in panel B) was measured. These have been compared with the IgA binding (▪ in panel A) and cell surface staining (▪) observed in quiescent HMCs. The results shown are means ± SEM of three different experiments performed in triplicate on a single HMC line. Activation resulted in enhanced IgA binding by HMC and up-regulation in HLA class I expression with induction of HLA class II synthesis; however, we were still unable to detect CD89 synthesis by HMCs. Similar results were obtained with PMA, LPS, TNF-α, and IL-6. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 7 Agarose gel electrophoresis of RT-PCR products derived from HMC (A) and PMA-activated U937 cells (B). cDNA were synthesized using AMV reverse transcriptase, followed by 30 cycles of amplification with Taq DNA polymerase using nine different human FcαR1-specific primer combinations: lane 1 (#1/#6), 2 (#2/#5), 3 (#3/#4), 4 (#2/#6), 5 (#2/#4), 6 (#3/#6), 7 (#3/#5), 8 (#1/#5), and 9 (#1/#4;A, upper panels) and GAPDH primers (B, lower panels). Only one primer pairing (#3/#4) generated the expected PCR product in HMCs, while all nine primer combinations generated the expected bands when RNA from PMA-activated U937 cells was used. This pattern of mRNA expression was consistent across each of the five primary HMC lines and did not change following mesangial activation. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 8 Diagrammatic representation of the FcαR1 mRNA transcripts detected in HMCs (A) and PMA-activated U937 cells (B). The upper panel represents wild-type (WT) FcαR1 transcript with the position of the six primers used in the PCR indicated by arrows. The three transcripts detected in HMCs exhibited partial identity with those found in PMA-activated U937 cells, although there were marked differences in both the 5′ and 3′ coding sequences as no PCR product could be generated when primers #1, #2, #5, and #6 were used. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 9 Northern blot analysis of CD89 mRNA (upper panel) expression by PMA-activated U937 cells and HMC cultured in medium alone (lane A), or in the presence of PMA (10-7 mol/L; lane B), LPS (1 μg/mL; lane C), IFN-γ (5 ng/mL; lane D), TNF-α (5 ng/mL; lane E) or IL-6 (100 U/mL; lane F). For these experiments, HMCs were allowed to grow to confluence and then rendered quiescent for 72 hours prior to incubation for 24 hours in test medium. The same blot was stripped and rehybridized with a cyclophilin-specific cDNA probe, to show equivalent RNA loading (lower panel). We were unable to demonstrate the presence of any homologous transcripts in HMCs; however, two CD89 hybridizing transcripts of 2.8 kb and 2.6 kb were consistently observed with PMA-activated U937 cells. Kidney International 2000 57, 1936-1948DOI: (10.1046/j.1523-1755.2000.00043.x) Copyright © 2000 International Society of Nephrology Terms and Conditions