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*
Immunonephrology Laboratory, Department of Research, University Hospital, Basel, Switzerland;
Department of Medicine, University Hospital, Geneva, Switzerland;
Blood Transfusion Center, University Hospital, Lausanne, Switzerland; and
§
Zentrallaboratorium Blood Donor Service, Bern, Switzerland
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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paraprotein and depletion of the early
components of the classical pathway of complement. The IgG
paraproteins were monomers with a normal structure, and with no
evidence for aggregation, as assessed by electron microscopy and
ultracentrifugation. Both heavy and light chains were of normal
molecular size (SDS-PAGE), and the paraproteins were not heavily
glycosylated. However, the paraproteins from all three patients had
unusual features that included abnormal behavior on gel filtration
chromatography and a heavy chain of high pI. When analyzed by fast
protein liquid chromatography (Superdex 200), elution of the
paraproteins was retarded, particularly when the ionic strength was
increased. This retardation was partially reversed in 20% alcohol, and
fully reversed in 6 M guanidine-HCl. Neither anti-C1 inhibitor nor
anti-C1q autoantibodies were found in any of the patients’ sera.
However, the paraproteins bound to the globular heads of C1q at normal
ionic strength. They activated C4 in normal human serum, but not in
C1q-deficient serum. Activation led to the formation of C1s-C1
inhibitor complexes. Taken together, the data suggest that the unusual
paraproteins have the capacity to bind C1q, which then leads to
activation of C1. The ability of these paraproteins to activate C1, in
spite of their being soluble monomers, is likely to be related to their
unique physicochemical features. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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paraproteins and low levels of the early components of the
classical complement pathway (C1q,r,s, C4, and C2). In the present
study, we analyzed the paraproteins of these two patients and of an
additional patient with the same syndrome. We found some common
features that may 1) explain specific interactions with complement, and
2) be related to the clinical syndrome. |
Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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All three patients investigated had recurrent panniculitis, an
IgG1 paraprotein (Table I
), and
hypocomplementemia (Table II). The
patients had neither angioedema nor evidence for SLE. The histology of
the panniculitic lesions showed nonspecific inflammatory infiltrates
with fat cell necrosis, but no vasculitis. All three were classified as
having Weber-Christian disease. The detailed clinical history of two of
the patients has been described by Pascual (patients B and C)
(11). Patient B had one more panniculitis flare, which
improved with steroids. He developed multiple myeloma 2 yr later and
died shortly afterward. Patient C had one major flare of panniculitis,
and was lost to follow-up 3 yr later. Patient F had three flares of
panniculitis with mediastinal involvement between 1994 and 1999. The
symptoms improved rapidly with steroids on each occasion. In 1999, he
had an acute myocardial infarction and died 48 h later. None had
plasmapheresis.
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The sera of the three patients were kept at -74°C until use. As controls we used the serum of a normal individual as well as six sera from patients with IgG myeloma and one further serum with an IgG paraprotein and plane xanthomatosis. These control patients had no history of panniculitis. In two of the myeloma patients, the IgG paraprotein was a cryoglobulin. Control sera containing anti-C1 inhibitor autoantibodies (kindly donated by Dr. Wüthrich, Zürich, Switzerland) and anti-C1q autoantibodies were stored at -74°C as well.
Complement protein concentrations were measured by radial immunodiffusion or nephelometry using standard techniques (12, 13). CH50 and AP50 using guinea pig cells were conducted as described by Mayer (14) and Platts-Mills (15, 16).
Cryoglobulins were measured as reported by Trendelenburg (17). Immunofixation electrophoresis was conducted as described by Alper (18).
Anti-C1 inhibitor autoantibodies
The anti-C1 inhibitor autoantibody ELISA was conducted as
described by Alsenz (9), with a slight modification. In
short, the wells (Maxisorp Nunc Immuno plates, Roskilde, Denmark) were
coated with saturating amounts of C1 inhibitor (Berinert HS from
Centeon Pharma) in sodium hydrogen carbonate buffer, pH 9.6, overnight
at room temperature. After washing, the sera were 1/25 diluted in PBS
and incubated for 1 h at 37°C. Bound IgG was detected with
biotinylated monoclonal mouse IgG anti-human 
-chain (Southern
Biotechnology Associates, Birmingham, AL) and streptavidin-HRP (Jackson
ImmunoResearch, West Grove, PA). The serum of a patient with known
anti-C1 inhibitor autoantibodies served as a positive control.
Values below the strongest positive signal of the 47 normal blood
donors were regarded as a negative test result.
Anti-C1q autoantibodies were determined using the method described by
Siegert (19). In short, ELISA wells (Maxisorp Nunc Immuno
plates) were coated overnight with 1 µg/well of C1q (Calbiochem, La
Jolla, CA) in sodium hydrogen carbonate buffer, pH 9.6, at room
temperature. After washing, the sera were incubated for 1 h at
37°C 1/25 diluted in PBS- Tween 0.05% containing 1% FCS (PBSTwFCS)
and 1 M NaCl. Bound IgG was detected with biotinylated monoclonal mouse
IgG anti-human -chain (Southern Biotechnology Associates)
diluted in PBSTwFCS and 1 M NaCl and streptavidin-HRP (Jackson
ImmunoResearch). After initial determination of the positivity for
anti-C1q autoantibodies, the serum of a patient with
hypocomplementemic urticarial vasculitis was established as reference
serum with an arbitrary value of 1000 U/ml. Titers lower than 80 U/ml
were considered to be undetectable. Forty-seven normal blood donors
were tested, from which two (4.3%) were positive
(20).
Low affinity binding of serum and of purified monomeric IgG fractions
to C1q was investigated by varying the amount of NaCl that was added to
the PBSTwFCS in the first incubation step. Furthermore, the results
obtained with the biotinylated monoclonal mouse IgG anti-human
-chain as second Ab were confirmed using biotinylated, polyclonal
goat anti-human -chain Abs (Calbiochem).
Abs directed to the collagen-like-region (CLR) of C1q (anti-CLR/C1q) were measured using pepsin-digested C1q prepared as reported by Reid (21). C1q was dialyzed overnight against freshly prepared 0.1 M sodium acetate, pH 4.45, at 4°C, and then incubated with pepsin (Sigma, St. Louis, MO) (1:30 v/v) for 20 h at 37°C. After centrifugation (30,000 x g at 4°C), the supernatant was applied to FPLC-Superdex 200 HR 10/30 (Amersham Pharmacia Biotech, Piscataway, NJ), and the first peak containing the CLR of C1q was pooled and used for the coating of the ELISA plates. The sera and IgG samples were diluted in PBSTwFCS and tested for the presence of anti-CLR/C1q.
Abs binding to the globular region of C1q (GR) were determined using collagenase-digested C1q, as reported by Wisnieski (22). The digest was applied to FPLC-Superdex 200 HR 10/30 (Amersham Pharmacia Biotech), and the third peak containing the GR of C1q was pooled and used for the coating of the ELISA plates. The sera and IgG samples were diluted in PBSTwFCS and tested for the presence of anti-GR/C1q with the same technique as mentioned above.
IgG purification
The sera were applied on protein G-Sepharose 4B (Sigma) and IgG eluted from the column using 0.1 M glycine HCl, pH 2.8. The pH of the fractions was immediately neutralized with 1 M Tris buffer, pH 9. For the separation of the paraproteins of the patients F, B, and C, the IgG-containing fractions were additionally applied to a Superdex 200 HR 10/30 gel filtration column (FPLC; Amersham Pharmacia Biotech) using a high salt buffer (PBS + 1 M NaCl). The fractions with the paraproteins were pooled, dialyzed against PBS, and concentrated with Microsep 30 K (Pall Filtron).
Heat-aggregated IgG (HAGG) was produced by heating normal human IgG to 63°C for 30 min. After being cooled in an ice bath, the proteins were precipitated with sodium sulfate, resuspended in PBS, and additionally dialyzed overnight at 4°C against PBS (final concentration of 0.4 mg/ml).
Purification of -heavy chains
The paraprotein of patient F and control normal human IgG were reduced, and the heavy chains were separated, as described by Fleischman (23). However, for the separation of the heavy chains by gel filtration, we used a Superdex 200 HR 10/30 with FPLC system (both Amersham Pharmacia Biotech) and 6 M guanidine-HCl as a dissociating buffer. Gel filtration was conducted twice, and the pooled heavy chain-containing fractions were dialyzed overnight against PBS at 4°C. The purity of the heavy chain was assessed by the lack of light chain on SDS-PAGE (24). The heavy chain-containing solutions were applied on the next day (no freezing) to the anti-C1q autoantibody ELISA without further addition of NaCl in the first incubation step.
Bidimensional gel electrophoresis of the paraproteins was conducted, as described by Tissot (25). The paraproteins were reduced, denatured, and separated in the first dimension by isoelectric focusing, and in the second dimension by SDS-PAGE. The polypeptides were silver stained (high m.w. proteins at the top, and acid proteins to the left).
Glycosylation of the paraproteins
In a first step, the unreduced Igs were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL; Amersham, Arlington Heights, IL); glycosylated proteins were revealed using the DIG glycan/protein double labeling kit (Boehringer Mannheim, Mannheim, Germany), according to the manufacturer’s indications.
Gel filtration chromatography
Gel filtration analysis of sera or purified Ig was performed using a FPLC system with a Superdex 200 HR 10/30 (cross-linked agarose and dextran) or a HiPrep16/60 Sephacryl S-300 (cross-linked copolymer of allyldextran and N,N-methylenebisacrylamide) column (Amersham Pharmacia Biotech) using PBS as a standard buffer. For further investigations, the running buffer was changed to PBS + 1 M NaCl, 50% PBS diluted in water, 20% ethanol in PBS, or 6 M guanidine-HCl.
The presence and relative amount of IgG in the gel filtration fractions were analyzed by the comparison of reduced and unreduced samples on SDS-PAGE
Ultracentrifugation.
To investigate whether the paraprotein of patient F is aggregated, we ultracentrifuged the serum for 2 h at 200,000 x g. Then it was applied to FPLC gel filtration, and the peak heights of the IgM, IgG, and paraprotein peaks were compared with their height before ultracentrifugation. To avoid variations due to small differences in the applied amounts of serum, the peak heights were expressed as a ratio to the height of peak 47, which contains small proteins (<1.35 kDa) or peptides. In the same way, we analyzed the loss of IgG and IgM in normal human serum (NHS) and in the serum of patients B and C.
Electron microscopy.
The paraproteins of patients B, C, and F were visualized by transmission electron microscopy and compared with normal human IgG and heat-aggregated normal human IgG. For the negative stain, purified IgG was adsorbed for 2 min to glow-discharged parlodion carbon-coated grids. After washing, all samples were stained with 0.75% uranylformate (pH 4.2). All samples were observed in a Zeiss EM 910 electron microscope operated at 80 kV acceleration voltage.
C4 consumption assay.
Complement-activating capacity of the different paraproteins was studied using a C4 hemolytic assay, as described previously (26). In short, washed sheep erythrocytes were coated with rabbit IgM anti-sheep erythrocytes (Amboceptor 6000) in barbitone-buffered saline for 20 min at 37°C. In a preincubation step, NHS 1/10 diluted in barbitone-buffered saline was incubated with different amounts of IgG preparations for 30 min at 37°C. The preincubation was stopped on ice and the mixture further diluted with barbitone-buffered saline to 1/100. Then, 20 µl of this mixture was added to the IgM-coated sheep erythrocytes together with 15 µl of undiluted guinea pig C4-depleted serum (Calbiochem) and incubated for 30 min at 37°C. Hemolysis was stopped on ice, and unlysed erythrocytes were removed by centrifugation (10 min, 1000 x g at 4°C). A total of 300 µl of the supernatants was applied in duplicates to a microtiter plate (Maxisorp Nunc Immuno plates), and the OD was measured at 405 nm. Each hemolytic step was done in duplicate, and the remaining hemolytically active C4 was calculated out of a standard curve with the diluted NHS used in the assays and guinea pig C4-depleted serum. The C4 consumption of the three paraproteins from the patients F, B, and C were compared with normal polyclonal IgG and with the IgG from three of six arbitrarily selected control myeloma patients. Aggregation of the purified Igs as a source of complement activation was minimized by a direct use of the preparations after purification and without further concentration after the gel filtration or by ultracentrifugation of the IgG preparations (1 h, 200,000 x g at 4°C).
In a modification of the assay described above, we replaced the NHS with human C1q-deficient serum or C1q-deficient serum + purified C1q (5 µg C1q/µl C1q-deficient serum) (both from Calbiochem). In the latter case, reassociation of the C1 complex was allowed for 30 min at 37°C before the IgG preparations were added.
C1s-C1 inhibitor complexes were measured by ELISA. Microtiter plates
(Maxisorp Nunc Immuno plates) were coated overnight at room temperature
with 0.6 µg/well purified, polyclonal goat anti-C1s -globulins
(Quidel, San Diego, CA) in sodium carbonate buffer and blocked with 2%
BSA PBS. After washing, the sera were diluted 1/400 in PBS and
incubated for 1 h at 37°C. Bound C1s-C1 inhibitor complexes were
detected with biotinylated, polyclonal goat anti-C1 inhibitor
-globulins (Quidel) and streptavidin-HRP (Jackson ImmunoResearch).
The standard curve was established using different dilutions of NHS
preincubated for 1 h at 37°C with HAGG (1 µg/µl NHS), which
had been shown to lead to a maximal formation of C1s-C1 inhibitor
complexes.
For the investigation of the complement-activating capacity of the paraproteins of patients B, C, and F compared with those of three myeloma patients, 5 µl NHS was incubated with varying amounts of IgG for 1 h at 37°C. The reaction was stopped on ice and the sera diluted in PBS to a final dilution of 1/400 before they were applied on the ELISA.
The effects of the paraproteins on the formation of C1s-C1 inhibitor complexes were tested by modifying the Quidel C1-Inhibitor Enzyme Immunoassay (Quidel). The biotinylated activated C1s was preincubated with normal polyclonal IgG; IgG preparations of the patients F, B, and C; or polyclonal goat anti-C1s IgG (Quidel) for 15 min at 37°C before mixing with purified C1 inhibitor (Berinert HS from Centeon Pharma). The final concentrations of C1 inhibitor specimens were 0.0055 mg/ml. The further steps were conducted according to the kit protocol.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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. All three patients had a depletion of the early components of the
classical pathway (Table II). Analysis of the paraproteins by gel filtration
To characterize the paraproteins and exclude the presence of
aggregates, we started to analyze the serum of the patients by gel
filtration using FPLC-Superdex 200 HR 10/30. The analysis of the serum
of patient F conducted with PBS as running buffer is shown in Fig. 1. In contrast to NHS, serum of patient F
had a low IgG, but an additional peak between 17 and 1.35 kDa. Analysis
of this protein peak by immunofixation and SDS-PAGE demonstrated that
it corresponded to the paraprotein (IgG) with an apparent size of
150 kDa. After reduction, the IgG split into the expected heavy and
light chains (50 and 25 kDa). Thus, the paraprotein behaved unusually
by being retarded on Superdex gel filtration. This retardation was also
seen on HiPrep16/60 Sephacryl S-300 gel filtration (data not shown).
Several different running buffers were used to see whether we could
abolish this retardation. PBS + 1 M NaCl led to a further retardation
with elution in the range of small peptides (Fig. 1). PBS/water (1:1)
also increased the retardation slightly. Reduction of the pH of PBS to
4 had no influence on the elution characteristics. With PBS + 20%
ethanol as a running buffer, the paraprotein was eluted together with
albumin. Finally, 6 M guanidine-HCl as a running buffer blocked the
retardation completely (Fig. 2), and the
paraprotein was eluted in the same fraction as normal IgG.
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). In patient B, the paraprotein was
eluted together with albumin when PBS was used as running buffer. PBS +
1 M NaCl led to an elution in the range of small proteins (i.e.,
retardation, but less pronounced than in patient F). In patient C, the
paraprotein eluted similarly to normal IgG (running buffer PBS), but
was significantly retarded if PBS + 1 M NaCl was used (i.e., eluted
with albumin). In both patients (B and C), PBS/water (1:1) had no
effect on the elution pattern (data not shown). Again 6 M guanidine-HCl
blocked the retardation completely.
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Because of this unexpected retardation phenomenon on gel filtration, the initial question, as to whether the paraproteins were forming aggregates, had to be investigated by a different technique.
Ultracentrifugation
The loss of the paraprotein in the serum of patient F after ultracentrifugation (20% after 2 h, 200,000 x g) was similar to the loss of IgG in the same serum (15%) or in NHS (16%), but less pronounced than the loss of the IgM in serum of patient F and NHS (38% and 34%, respectively). Similar observations to those made in patient F were made in the sera of patients B and C.
Electron microscopy
The purified monoclonal IgG of the three patients were analyzed
by electron microscopy using normal IgG and normal HAGG as controls.
The monoclonal Igs were dissociated, and never formed any aggregates,
which was in clear contrast to Ig, which had been heated at 63°C
(Fig. 4). The IgG paraproteins had the
expected morphological characteristics of IgG (with distinguishable
F(ab')2 and Fc portions) and were microscopically
not different from normal polyclonal IgG.
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Having established that aggregation was not the explanation for
the retardation on the gel filtration and the hypocomplementemia of the
patients, we analyzed the charge of the three paraproteins isolated by
affinity chromatography on protein G. The monoclonal heavy chains from
patients presenting monoclonal gammopathies were easily differentiated
from polyclonal heavy chains, according to their different
two-dimensional electrophoretic patterns (25) (Fig. 5). Monoclonal heavy chains showed charge
microheterogeneity, whereas polyclonal heavy chains were highly
heterogeneous and were resolved as unspotted diffuse zones extending in
a zone corresponding to pI from 5.5 to more than 9. When the purified
IgG fractions from the three patients were analyzed, the spots
corresponding to the monoclonal -chains were not observed onto the
gels, as exemplified for patient F (Fig. 5A), indicating
that their charges were highly basic, contrasting with the control
myeloma patient depicted in Fig. 5B, whose monoclonal
-chain appeared as a set of well-resolved spots in the area of the
gel corresponding to a pI of about 7 (Fig. 5B). No apparent
particular electrophoretic properties were evidenced when analyzing the
monoclonal Ig light chains from the three patients and from the control
myeloma patients. Monoclonal light chains from patients and control
myeloma patients disseminate in more than one spot, and were
characterized by different pI. These patterns were clearly different
from that of polyclonal Ig light chains (Fig. 5C).
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Finally, heavy glycosylation of the three paraproteins as a possible explanation for the retardation described above was tested on immunoblots using a DIG glycan/protein double labeling kit. However, using that assay, the glycosylation of the paraproteins of patients F, B, and C was not found to be different from the glycosylation of normal polyclonal IgG (data not shown).
Paraprotein binding to complement proteins
Because similar complement profiles as found in these three patients were described in patients with either anti-C1 inhibitor or anti-C1q autoantibodies, we tested for the presence of both types of autoantibodies. The three sera were negative for anti-C1 inhibitor autoantibodies.
The possible binding of the paraproteins to C1q was investigated using
C1q-coated plates, as done for the anti-C1q autoantibody ELISA. In
high salt buffer, i.e., the classical anti-C1q autoantibody assay
(19), no binding of Ig to C1q could be seen in the sera of
patient F and C, whereas there was a clear signal in the serum of
patient B. Lowering the ionic strength below the physiological value
allowed the detection of specific binding of IgG to C1q in sera F (Fig. 6) and C as well. This signal was not
influenced by varying the pH between 6 and 9. Replacing the detecting
monoclonal mouse anti-human heavy chain by a polyclonal Ab did not
modify the results. Testing all fractions eluted from FPLC-Superdex
directly, the IgG binding to C1q was shown to be restricted to the IgG
paraprotein in all three sera. Interestingly, under these conditions,
the specific C1q binding of the two paraproteins F (Fig. 7) and C was evident even at
physiological ionic strength, suggesting that this binding was hindered
in whole serum and unmasked when the eluted FPLC fractions were tested
separately. However, such weak interactions with C1q were not entirely
specific for the paraproteins investigated in this study. In low ionic
strength buffer, three of the six control sera of myeloma patients were
also showing some binding, although two of these three controls that
were positive had strong cryoglobulinemia (>1 mg/ml).
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). When GR of C1q were used as the
Ag, there was no drop of the signal as seen for anti-C1q
autoantibodies (Fig. 9). These results
indicated the globular heads of C1q as the binding site for the
paraproteins.
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).
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The capacity of the three paraproteins to activate complement in
vitro was investigated by a C4 hemolytic assay. Whereas normal human
IgG only slightly consumed C4 (<10%) in concentrations up to 50 mg
IgG/ml NHS, the three paraproteins showed a dose-dependent consumption
of C4 in NHS. In direct comparison, the paraprotein of patient B caused
the strongest C4 loss (Fig. 11). Three
IgG paraproteins purified from the serum of myeloma patients produced
only a minimal loss of C4.
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). In addition, in all three
patients, the preincubation of the paraproteins with NHS led to a
dose-dependent formation of C1s-C1 inhibitor (Fig. 13).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-chains. Finally, the
paraproteins bound the globular heads of C1q, unlike anti-C1q
autoantibodies, and activated the classical pathway of complement via
C1q despite being monomers in solution. The retardation by gel filtration was evident using different gel matrices and was not abolished by high salt concentrations. Thus, charge-charge interactions between the paraproteins and the gel matrix could not be responsible for these retardations, although the highly cationic charge of the heavy chains of all three paraproteins might have suggested such interactions. In contrast to that, the paraproteins of patient F and B were even further retarded by a high salt buffer, and in patient C, the retardation of the paraprotein could only be observed under high salt conditions. In addition, the low salt buffer enhanced the retardation of the paraprotein of patient F. These observations suggest a more complex interaction between the paraproteins and the gel filtration matrix, which could be completely prevented only by a strongly dissociating buffer such as 6 M guanidine-HCl. We analyzed six other paraproteins from myeloma patients, and in addition the paraprotein of a patient with plane xanthomatosis and low C1q but otherwise normal complement, which all eluted similarly to normal polyclonal IgG. We have not been able to find a description of a similarly retarded paraprotein in the literature. It will be interesting to see whether this unique retardation profile for a paraprotein, enhanced by high salt, will be found exclusively in patients with the clinical syndrome of recurrent panniculitis and hypocomplementemia.
The IgG paraprotein of all three patients bound C1q. Whereas the binding of the paraproteins to C1q could be inhibited by a high salt buffer in patients F and C, this was not possible in patient B. In the most often used anti-C1q autoantibody assay (19), this patient would have been declared as having anti-C1q autoantibodies. However, anti-C1q autoantibodies are described to bind to the CLR of C1q. In contrast to that, we were able to demonstrate that the paraprotein of patient B, as those of patients F and C, bound to the GR of C1q and not to the CLR. It can be speculated that the strong binding of paraprotein B to C1q was responsible for the stronger complement activation compared with the two other paraproteins investigated in this study.
It was striking that the binding to C1q, although almost undetectable in serum for two patients, leads to an efficient activation of C1 and cleavage of C4. According to our findings, this is not due to an impaired formation of C1s-C1 inhibitor complexes. However, considering the high concentration of the paraproteins in vivo compared with the concentration of C1q, it seems to be likely that already the weak affinity of the paraproteins to C1q was sufficient for complement activation.
Although three of six control myeloma paraproteins had a similar low affinity binding to C1q as the paraproteins of patients F and C, they did not activate complement. It is possible that the unique biochemical properties of these paraproteins were responsible for this apparent discrepancy. The strongly positively charged heavy chains in all three patients could explain the measured complement activation. Polycationic molecules have been described as reacting with C1q to induce complement activation via the classical pathway. At the same time, they are known to inhibit the assembly of the alternative pathway C3 convertase by interfering with the binding of C3b to factor B, and they exert an inhibitory effect on C7, C8, and C9 (27). These findings could explain the profile of complement components found in our three patients, in whom the early classical pathway components were depleted, but C3, the AP, and the lytic pathway were normal.
The finding of a monomeric IgG capable of binding C1q and trigger complement activation in the fluid phase would be unique. For this reason, we set out to see whether the paraproteins of the three patients had the property to associate and form polymers in the fluid phase. Such associations could be shown neither by ultracentrifugation nor by electron microscopy. In particular, the general structures of the paraproteins appear completely normal on electron-microscopic pictures.
The link between an ongoing complement activation in these patients and the occurrence of recurrent panniculitis, if there is one, remains speculative. Very probably the hypocomplementemia is due to the presence of the abnormal paraprotein. However, the clinical history of all three patients suggests the need of an additional triggering mechanism such as a trauma or an infection to cause panniculitis. Adipocytes are known to produce all the components of the alternative pathway (C3, factor B, and factor D) (28, 29, 30). Furthermore, adipocytes had been shown to fix C1q at their surface (31). Thus, it may be that, in the presence of the paraproteins, at the time of a minimal change in the local synthesis or binding capacity of complement proteins by adipocytes, the cascade is triggered and responsible for local inflammation.
In conclusion, we present three patients with recurrent panniculitis who had highly cationic IgG paraproteins with abnormal retardation on gel filtration. We demonstrated that these soluble monomeric IgG paraproteins directly activated the classical complement pathway. It is likely that they were responsible for the hypocomplementemia of these patients. Our findings provide further evidence that the entity of recurrent s.c. and visceral panniculitis, paraproteinemia, and hypocomplementemia is a unique and well-defined syndrome.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marten Trendelenburg, Immunonephrology Laboratory, Department of Research, University Hospital, Basel Hebelstrasse 20, CH-4031, Basel, Switzerland. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; CLR, collagen-like region; FPLC, fast protein liquid chromatography; GR, globular region; HAGG, heat-aggregated IgG; NHS, normal human serum.
Received for publication March 22, 1999. Accepted for publication October 6, 1999.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-globulins. Biochem. J. 88:220.
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= polyclonal Ig light chains; 
95%), indicating that the paraproteins did not bind
the CLR of C1q (CLR/C1q).
