Ann Hematol DOI 10.1007/s00277-006-0158-5
ORIGINAL ARTICLE
Polyclonal IgG4 hypergammaglobulinemia associated with plasmacytic lymphadenopathy, anemia and nephropathy Emmanuelle Boulanger & Vincent Fuentes & Véronique Meignin & Béatrice Mougenot & Sylvaine Labaume & Valérie Gouilleux-Gruart & Michel Cogné & Pierre Aucouturier & Jean-Pierre Clauvel & Pierre Ronco & Kaiss Lassoued Received: 27 September 2005 / Accepted: 7 June 2006 # Springer-Verlag 2006
Abstract Marked polyclonal immunoglobulin (Ig)G4 hypergammaglobulinemia has exceptionally been reported. Here we report on two Algerian patients who presented a syndrome characterized by anemia, plasmacytic lymphadeE. Boulanger : J.-P. Clauvel Department of Clinical Immunology, Hôpital Saint-Louis, AP-HP, Paris, France V. Fuentes : V. Gouilleux-Gruart : K. Lassoued Inserm E0351, Department of Immunology, Hôpital Nord, Amiens, France V. Meignin Laboratory of Pathology, Hôpital Saint-Louis, AP-HP, Paris, France B. Mougenot Laboratory of Pathology, Hôpital Tenon, AP-HP, Paris, France S. Labaume IFR105, Laboratory of Immunopathology, Hôpital Saint-Louis, Paris, France M. Cogné UMR CNRS 6101, Laboratory of Immunology, CHU, Limoges, France P. Aucouturier Laboratory of Immunochemistry, Hôpital Tenon, AP-HP, Paris, France P. Ronco Department of Nephrology, Hôpital Tenon, AP-HP, Paris, France E. Boulanger (*) Service d’Immunopathologie Clinique, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75 475 Paris Cedex 10, France e-mail:
[email protected]
nopathy, renal manifestations, and a marked polyclonal IgG4 hypergammaglobulinemia leading to a hyperviscosity syndrome in one case. The IgG4-expressing cell percentage was significantly increased in the peripheral blood lymphocytes collected from the two patients upon diagnosis. Moreover, in contrast with normal sera, both patients’ sera significantly increased the percentage of IgG4-expressing cells when incubated with CD40-stimulated normal B lymphocytes. Similar effects were obtained with the culture supernatants of the patients’ activated T cells. Antiinterleukin (IL) 4 and/or anti-IL-13 antibodies were unable to antagonize the IgG4 production. IL-4 and IL-13 serum concentrations were found to be normal in the two patients. The increased IgG4 production was found to be mediated by soluble factor(s), most probably secreted by activated T cells, which did not require the signal transducer and activator of transcription 6 signaling pathway. Keywords Hypergammaglobulinemia . IgG4 . Hyperviscosity . Plasmacytic lymphadenopathy . Proteinuria
Introduction Immunoglobulin (Ig)G4 immunoglobulins correspond to the less abundant IgG subclass in the serum and are undetectable in 30% of healthy individuals [3]. Polyclonal hypergammaglobulinemia is usually associated with liver disorders, autoimmune diseases, chronic infections, and hematological malignancies [5]. Patients with massive polyclonal hypergammaglobulinemia exceptionally develop hyperviscosity syndrome [2]. Moreover, in most cases, several immunoglobulin (Ig) isotypes are involved. Polyclonal increase in the IgG4 subclass has been observed in allergic disorders [1] and parasitic infections [8], as well as
Ann Hematol
in autoimmune pancreatitis [7], multicentric Castleman disease [10], vasculitis, respiratory diseases, and lymphoproliferative disorders [24]. However, IgG4 levels above 15 g l−1 have never been reported so far. We report here on two patients with a marked polyclonal IgG4 hypergammaglobulinemia associated with lymphoid organ enlargement, anemia, and renal manifestations. The increased IgG4 production appeared to be mediated by some unidentified signal transducer and activator of transcription 6 (Stat6)independent soluble factor(s) secreted by activated T lymphocytes and distinct from interleukin (IL) 4 and IL-13.
Materials and methods Patients and controls The two patients were admitted at the Hôpital Saint-Louis. Their clinical and biological data have been collected prospectively (see below and Table 1). Peripheral blood mononuclear cells (PBMCs) (and sera) were collected from both patients upon diagnosis and from six healthy individuals and purified by Ficoll–Hypaque gradient centrifugation. Adherent cells were removed after 1 h incubation at 37°C in plastic flasks. Fresh T (E+) and B (E−) peripheral blood lymphocytes (PBLs) were separated by sheep red cell rosetting, yielding a high purity level. Normal B cells were also purified from the spleen of transplant organ cadaveric donors. Serum Ig and cytokine levels Serum IgM, IgG, and IgA concentrations were determined by nephelometry and serum IgE by radioimmunoassay. Serum IgG subclass levels were determined as previously described [3] and using a commercial kit (Binding Site, Birmingham, UK). Enzyme-linked immunosorbent assay (ELISA) methods were used to measure the serum levels of IL-2, IL-4, IL-7, IL-13, macrophage inflammatory protein (MIP)-1α, RANTES (R&D Systems, Minneapolis, MN, USA), and human growth hormone (hGH; Abazyme, Needham, MA, USA).
antibodies to human insulin-like growth factor I (IGF-I) were also used (Leinco Technologies Inc., Saint Louis, MO, USA). In each experiment, the percentage of Ig-expressing cells was assessed by indirect immunofluorescence (IF) analysis performed on centrifuged preparations of air-dried PBL either freshly purified or analyzed after in vitro culture. Cell culture experiments All cultures were performed in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with heat-inactivated 10% fetal calf serum, 2 mM L-glutamine, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin. B and T cells purified from each patient or healthy individual were cocultured for 6 days at a ratio of 2:1 into 24-well plates as triplicates in the presence of anti-CD3 (0.25 μg ml−1) and anti-CD28 (0.5 μg ml−1) mAb or with phytohemagglutinin (PHA) at 5 μg ml−1. In other experiments, B cells were cultured for 6 days in the presence of IL-10 (Peprotech, Le Perray-en-Yvelines, France) at 10 ng ml−1, with irradiated CD32 (FcγRII)-expressing fibroblasts [4] and anti-CD40 mAbs (1 μg ml−1), or with irradiated CD40 ligand (CD40L)-expressing fibroblasts. In each case, 50 μg ml−1 anti-IL-4- and/or 50 μg ml−1 anti-IL-13neutralizing mAbs, or isotype-matched mAbs, were added to the cell cultures. Normal B cells were cultured for 6 days with irradiated CD40L-expressing fibroblasts in the presence of IL-10 (10 ng ml−1). The serum obtained from each patient or healthy subject was added to the cell culture at a one-fourth dilution after IgG depletion on a protein G affinity column. After IgG depletion, the residual IgG4 concentrations were less than 0.1 g l−1. Neutralizing anti-IL-4 (50 μg ml−1) and anti-IL-13 (50 μg ml−1) mAbs, as well as neutralizing rabbit polyclonal Abs to human IGF-I (10 μg ml−1), were added to cell cultures. In other experiments, instead of serum, IgG-depleted culture supernatant of activated T cells obtained from each patient or healthy individual was added to the B-cell culture. Electromobility gel shift assay
Antibodies and indirect immunofluorescence assay Monoclonal antibodies (mAbs) against human γ1 and γ4 Ig heavy chains were obtained from Oxoid (Dardilly, France). Other mAbs included the antihuman μ (Clinisciences, Montrouge, France), anti-CD3 (Clinisciences), anti-CD13, anti-CD20, anti-CD23 and anti-CD28 (Beckman Coulter, Fullerton, CA, USA), anti-CD40 (American Type Culture Collection, Rockville, MD, USA), antihuman IL-4, and IL-13 (Diaclone, Besançon, France) antibodies. Rabbit polyclonal
Whole cell extracts were prepared from untreated patients’ PBL and from normal B lymphocytes cultured for 30 min in the presence or absence of IL-4 at 100 ng ml−1 (Eurocetus, Amsterdam, The Netherlands), or patients’ plasma or serum at 1:5 dilution. Cell extracts were used in bandshift assays as previously described [25]. The oligonucleotide 5′-GTCAACTTCCCAAGAACAGAA-3′ containing the Stat6-binding site of Iɛ gene promoter was 32 P-end-labeled and used as a probe.
Ann Hematol Table 1 Main clinical and biological features at the time of diagnosis Characteristics
Patient 1
Patient 2
Sex/Age (years) Lymphadenopathy/Splenomegaly Hemoglobin level (g dl−1) Leukocyte count (×109 l−1) Platelet count (×109 l−1) Serum C-reactive protein (mg l−1, N
