Clin. exp. Immunol. (1979) 35, 376-379.
Polymorphonuclear leucocyte fluorescence and cryoglobulin phagocytosis in systemic lupus erythematosus A. L0 S I TO & L. LORUS SO Haemodialysis Unit, General Regional Hospital, Perugia, Italy
(Received 31 July 1978) SUMMARY
Peripheral blood polymorphonuclear leucocytes (PMN) and cryoglobulins were isolated from patients with systemic lupus erythematosus (SLE). Fluorescent inclusions were found in PMN. Normal donor PMN were incubated with the sera and cryoglobulins from SLE patients. In most cases inclusions were observed after incubation. The high incidence of anti-IgG activity in phagocytosed cryoglobulins confirms the importance of the rheumatoid factor in phagocytosis of immune complexes. It is concluded that phagocytosis of cryoglobulins supports the suggestion that cryoglobulins are a subpopulation of immune complexes. INTRODUCTION Immunofluorescence studies in polymorphonuclear leucocytes (PMN) from patients with systemic lupus erythematosus (SLE) and other immune complex-mediated diseases have revealed cytoplasmic inclusions composed of immunoglobulins and complement components (Cats, Lafeber & Klein, 1975; Steffelaar, de Graaff-Reitsma & Feltkamp-Vroom, 1976; Lewis & Pegrum, 1977). These inclusions are considered to be in vivo phagocytosis of immune complexes. In vitro studies have revealed cytoplasmic inclusions in the PMN of normal donors after incubation with SLE sera and have also shown that HBsAg/anti-HBs immune complexes prepared artificially in a slight antibody excess are phagocytosed by normal PMN (Steffelaar et al., 1977). It has been demonstrated that cryoglobulins containing DNA/anti-DNA isolated from patients suffering from SLE and other conditions are immune complexes with a slight antigen excess (Wands et al., 1978). The present study was undertaken to investigate (1) the relationship between cytoplasmic inclusions and cryoglobulin composition in patients with SLE, and (2) whether phagocytosis of cryoglobulins occurs in vitro after incubation with normal donor PMN.
MATERIALS AND METHODS Subiects. Twelve SLE patients with degrees of disease activity and whose sera contained cryoglobulins were studied. The diagnosis was made according to the criteria proposed by the American Rheumatism Associations (ARA) (Cohen et al., 1971) and the ANA antibody test was positi e in all patients with a homogeneous pattern. All patients were treated with steroids. Twelve race-, age- and sex-matched subjects of the laboratory staff acted as controls. Cryoglobulins. For isolation, 20-30 ml blood were allowed to clot at 37°C for 3 hr. Serum was then separated by centrifugation at 2500 g for 20 min at 37°C and stored at 4°C for 6 days. The cryoprecipitate was separated by centrifugation at 3000 g for 30 min at 4°C and washed five times in cold phosphate buffered saline (PBS); it was then dissolved by adding one twentieth part of the original serum volume of 0-28 Ni sodium chloride, mixed for 2 hr at 37°C and, finally, an equal volume of distilled water was added. After checking for the absence of albumin by double diffusion, the precipitate was tested at 37°C with monospecific antisera (Behringwerke) for IgG, IgA, IgM Clq, C4, C4, C3PA and fibrinogen; the same method was used throughout.
Corre-,pondence: Dr A. Losito, via Pellini 4, 06100 Perugia, Italy. 0099-9104/79/030-0376$02.00 CC) 1979 Blackwell Scientific Publications
376
PMN and cryoglobulins in SLE
377
When found, IgG, IgA and IgM were measured by single radial immunodiffusion. Anti-gammaglobulin activity of the cryoglobulins was tested by agglutination of sheep red blood cells sensitized with rabbit gammaglobulins (CellognostBehringwerke). Complement components in sera were measured with single radial immunodiffusion. Polymorphonuclear leucocytes (direct test). 5 0 ml 5% dextran in 099% saline (mol. wt 70,000) (Baxter, Trieste, Italy) were added to 10 ml of fresh heparinized blood. Red blood cells (RBC) were allowed to sediment at 37 C for 45 min. The supernatant wvas resuspended in Hanks's balanced salt solution (HBSS) and centrifuged at 400 g for 15 min. The pellet was resuspended in 0 5 ml HBSS and layered onto the top of 1 5 ml lymphoprep (Pharmacia). After centrifugation at 400 g for 40 min, the interface containing the mononuclear cells was aspirated and the medium discarded. The pellet was resuspended in HBSS and spun at 200 g for 15 min. When the supernatant had been aspirated, ten drops of 2% paraformaldehyde solution in PBS and ten drops of HBSS were added to the pellet. After a further 5 min, the cells were centrifuged at 200 g for 10 min. The supernatant was aspirated and the cells were resuspended in five to seven drops of 5% gelatine solution in HBSS, counted in a Burker chamber and checked for viability by trypan blue dye exclusion. The cells, at a concentration ranging from 3-5 x 106 PMN/ml, were snap frozen in a gelatine capsule in liquid nitrogen. Contamination with mononuclear cells, platelets and RBC was negligible. The control, with blood collected in 5 0 mrv monoiodacetic acid (MIAA), pH 7-2, was always performed to block 'in vitro phagocytosis'. Phagocytosis in vitro (indirect test). Heparinized blood collected from normal donors was processed as above until PMN isolation; then four separate isolations were set up: (1) PMN with HBSS; (2) PMN with cryoglobulins; (3) PMN with patient serum (diluted 1: 5) and (4) PMN with normal donor serum (diluted 1: 5). The four tubes were put in a Matburn rotator at 370C for 30 min. After incubation, the cells were resuspended in HBSS and centrifuged for 15 min at 200 g. The supernatant was then discarded and the cells were fixed and processed as above. Immunofluorescence procedure. Cryostat sections 40 ,um were cut from the frozen gelatine capsules. The sections were airdried for 30 min and then incubated with rabbit monospecific antisera conjugated with fluorescin (Behringwerke) against human IgG, IgA, IgM, C3, C4 and Clq. For nuclear counter-staining, part of the sections were incubated with ethidium bromide 0-02 mg/ml solution in PBS (Calbiochem). After cooling, the slides were read with a fluorescence microscope (Reichert) with incident illumination by two independent observers. A total of at least 300 cells were screened for each sample.
RESULTS Table 1 shows the results of leucocyte fluorescence, incubation tests, cryoglobulins and serum complement in the twelve patients studied. None of the controls had leucocyte fluorescence and when normal cells were incubated with normal serum or HBSS no fluorescent inclusions were detected. The direct test performed with MIAA gave the same results as the cells collected with heparin only. Cryoglobulins
FIG. 1. Large cytoplasmic inclusions cryoglobulins). I)
seen
by immunofluorescence with anti-IgG (indirect test with
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Polymorphonuclear leucocyte fluorescence and cryoglobulin phagocytosis in systemic lupus erythematosus A. L0 S I TO & L. LORUS SO Haemodialysis Unit, General Regional Hospital, Perugia, Italy
(Received 31 July 1978) SUMMARY
Peripheral blood polymorphonuclear leucocytes (PMN) and cryoglobulins were isolated from patients with systemic lupus erythematosus (SLE). Fluorescent inclusions were found in PMN. Normal donor PMN were incubated with the sera and cryoglobulins from SLE patients. In most cases inclusions were observed after incubation. The high incidence of anti-IgG activity in phagocytosed cryoglobulins confirms the importance of the rheumatoid factor in phagocytosis of immune complexes. It is concluded that phagocytosis of cryoglobulins supports the suggestion that cryoglobulins are a subpopulation of immune complexes. INTRODUCTION Immunofluorescence studies in polymorphonuclear leucocytes (PMN) from patients with systemic lupus erythematosus (SLE) and other immune complex-mediated diseases have revealed cytoplasmic inclusions composed of immunoglobulins and complement components (Cats, Lafeber & Klein, 1975; Steffelaar, de Graaff-Reitsma & Feltkamp-Vroom, 1976; Lewis & Pegrum, 1977). These inclusions are considered to be in vivo phagocytosis of immune complexes. In vitro studies have revealed cytoplasmic inclusions in the PMN of normal donors after incubation with SLE sera and have also shown that HBsAg/anti-HBs immune complexes prepared artificially in a slight antibody excess are phagocytosed by normal PMN (Steffelaar et al., 1977). It has been demonstrated that cryoglobulins containing DNA/anti-DNA isolated from patients suffering from SLE and other conditions are immune complexes with a slight antigen excess (Wands et al., 1978). The present study was undertaken to investigate (1) the relationship between cytoplasmic inclusions and cryoglobulin composition in patients with SLE, and (2) whether phagocytosis of cryoglobulins occurs in vitro after incubation with normal donor PMN.
MATERIALS AND METHODS Subiects. Twelve SLE patients with degrees of disease activity and whose sera contained cryoglobulins were studied. The diagnosis was made according to the criteria proposed by the American Rheumatism Associations (ARA) (Cohen et al., 1971) and the ANA antibody test was positi e in all patients with a homogeneous pattern. All patients were treated with steroids. Twelve race-, age- and sex-matched subjects of the laboratory staff acted as controls. Cryoglobulins. For isolation, 20-30 ml blood were allowed to clot at 37°C for 3 hr. Serum was then separated by centrifugation at 2500 g for 20 min at 37°C and stored at 4°C for 6 days. The cryoprecipitate was separated by centrifugation at 3000 g for 30 min at 4°C and washed five times in cold phosphate buffered saline (PBS); it was then dissolved by adding one twentieth part of the original serum volume of 0-28 Ni sodium chloride, mixed for 2 hr at 37°C and, finally, an equal volume of distilled water was added. After checking for the absence of albumin by double diffusion, the precipitate was tested at 37°C with monospecific antisera (Behringwerke) for IgG, IgA, IgM Clq, C4, C4, C3PA and fibrinogen; the same method was used throughout.
Corre-,pondence: Dr A. Losito, via Pellini 4, 06100 Perugia, Italy. 0099-9104/79/030-0376$02.00 CC) 1979 Blackwell Scientific Publications
376
PMN and cryoglobulins in SLE
377
When found, IgG, IgA and IgM were measured by single radial immunodiffusion. Anti-gammaglobulin activity of the cryoglobulins was tested by agglutination of sheep red blood cells sensitized with rabbit gammaglobulins (CellognostBehringwerke). Complement components in sera were measured with single radial immunodiffusion. Polymorphonuclear leucocytes (direct test). 5 0 ml 5% dextran in 099% saline (mol. wt 70,000) (Baxter, Trieste, Italy) were added to 10 ml of fresh heparinized blood. Red blood cells (RBC) were allowed to sediment at 37 C for 45 min. The supernatant wvas resuspended in Hanks's balanced salt solution (HBSS) and centrifuged at 400 g for 15 min. The pellet was resuspended in 0 5 ml HBSS and layered onto the top of 1 5 ml lymphoprep (Pharmacia). After centrifugation at 400 g for 40 min, the interface containing the mononuclear cells was aspirated and the medium discarded. The pellet was resuspended in HBSS and spun at 200 g for 15 min. When the supernatant had been aspirated, ten drops of 2% paraformaldehyde solution in PBS and ten drops of HBSS were added to the pellet. After a further 5 min, the cells were centrifuged at 200 g for 10 min. The supernatant was aspirated and the cells were resuspended in five to seven drops of 5% gelatine solution in HBSS, counted in a Burker chamber and checked for viability by trypan blue dye exclusion. The cells, at a concentration ranging from 3-5 x 106 PMN/ml, were snap frozen in a gelatine capsule in liquid nitrogen. Contamination with mononuclear cells, platelets and RBC was negligible. The control, with blood collected in 5 0 mrv monoiodacetic acid (MIAA), pH 7-2, was always performed to block 'in vitro phagocytosis'. Phagocytosis in vitro (indirect test). Heparinized blood collected from normal donors was processed as above until PMN isolation; then four separate isolations were set up: (1) PMN with HBSS; (2) PMN with cryoglobulins; (3) PMN with patient serum (diluted 1: 5) and (4) PMN with normal donor serum (diluted 1: 5). The four tubes were put in a Matburn rotator at 370C for 30 min. After incubation, the cells were resuspended in HBSS and centrifuged for 15 min at 200 g. The supernatant was then discarded and the cells were fixed and processed as above. Immunofluorescence procedure. Cryostat sections 40 ,um were cut from the frozen gelatine capsules. The sections were airdried for 30 min and then incubated with rabbit monospecific antisera conjugated with fluorescin (Behringwerke) against human IgG, IgA, IgM, C3, C4 and Clq. For nuclear counter-staining, part of the sections were incubated with ethidium bromide 0-02 mg/ml solution in PBS (Calbiochem). After cooling, the slides were read with a fluorescence microscope (Reichert) with incident illumination by two independent observers. A total of at least 300 cells were screened for each sample.
RESULTS Table 1 shows the results of leucocyte fluorescence, incubation tests, cryoglobulins and serum complement in the twelve patients studied. None of the controls had leucocyte fluorescence and when normal cells were incubated with normal serum or HBSS no fluorescent inclusions were detected. The direct test performed with MIAA gave the same results as the cells collected with heparin only. Cryoglobulins
FIG. 1. Large cytoplasmic inclusions cryoglobulins). I)
seen
by immunofluorescence with anti-IgG (indirect test with
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