Clin. exp. Immunol. (1977) 28, 413-425.
Phagocytosis of immune complexes by polymorphonuclear leucocytes in patients with Felty's syndrome* E. R. HURD,t M. ANDRE I S & M. ZIFF§ Dallas Veterans Administration Hospital, Dallas, and The University of Texas Health Science Center, Dallas, Texas
(Received 29 November 1976) SUMMARY
The possible role of phagocytosis of circulating immune complexes by neutrophils in the production of the neutropenia of Felty's syndrome has been investigated. Normal neutrophils phagocytosed massive inclusions from the sera from twelve of fifteen patients with Felty's syndrome when incubated with these sera. Such inclusions were phagocytosed from only three of fifteen patients with seropositive RA who did not have Felty's syndrome. Normal neutrophils were more effective than patient neutrophils with regard to phagocytosis of inclusions from the patients' serum suggesting a defect in phagocytic function of Felty's neutrophils. The titre of granulocyte-reactive antinuclear antibodies did not appear to be related to the degree of neutropenia. The data suggest that phagocytosis of circulating immune complexes by neutrophils may interfere with the function of these cells in combating infection and also render them susceptible to removal from the circulation thus leading to the development of neutropenia.
INTRODUCTION Felty's syndrome consists of the triad of rheumatoid arthritis, leukopenia and splenomegaly (Felty, 1924). Patients with this syndrome may have frequent infections (De Gruchy & Langley, 1961; Barnes, Turnbull & Vernon-Roberts, 1971; Denko & Zumpft, 1962; Ruderman, Miller & Pinals, 1968; Hahn, Mayne & Kiely, 1963). They usually have high titres of rheumatoid factor and frequently, positive tests for antinuclear antibodies and LE cells (DeGruchy & Langley, 1961; Barnes et al., 1971; Denko & Zumpft, 1962; Ruderman et al., 1968; Hahn et al., 1963). The patients tend to have severe, though frequently inactive arthritis, subcutaneous nodules, lymphadenopathy, anemia, thrombocytopenia, manifestations of vasculitis such as chronic leg ulcers and peripheral neuropathy and an increased incidence of Sjogren's syndrome (DeGruchy & Langley, 1961; Barnes et al., 1971; Denko & Zumpft, 1962; Ruderman et al., 1968; Hahn et al., 1963). The leukopenia is due primarly to a neutropenia and is associated with an increase in myeloid activity in the bone marrow (DeGruchy & Langley, 1961). The etiology of the leukopenia has been obscure, although proposed mechanisms have included neutrophil-specific antinuclear antibodies (Faber & Elling, 1966), margination of the neutrophils (Vincent, Levi & Macqueen, 1974) and impaired release of neutrophils from the bone marrow (Brubaker, Johnson & Morris, 1974). The present study presents data which suggests another possible mechanism for the neutropenia, i.e. phagocytosis of circulating immune complexes by the circulating granulocytes. Presented in part before the 4th Pan American Congress of Rheumatology, Toronto, Ontario, Canada, June, 1974. t Clinical Scholar, Arthritis Foundation and previously, Clinical Investigator, Dallas V.A. Hospital, and Associate Professor of Internal Medicine, The University of Texas Health Science Center, Dallas. t Assistant Professor of Internal Medicine, The University of Texas Health Science Center, Dallas; Postdoctoral Fellow, The Arthritis Foundation. § Professor of Internal Medicine, The University of Texas Health Science Center, Dallas; Recipient, Research Career Award, US Public Health Service. Correspondence: Dr Eric R. Hurd, Department of Internal Medicine, The University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, U.S.A. *
413
414
E. R. Hurd, M. Andreis & M. Ziff MATERIALS AND METHODS
Patient and control population. Studies were performed on the blood taken from ten normal persons, fifteen with Felty's syndrome, fifteen with classical rheumatoid arthritis (RA) as defined by the ARA diagnostic criteria (Ropes et al., 1959; Cohen et al., 1971), but without evidence of splenomegaly or leukopenia, three with Sjogren's syndrome, two with rheumatoid vasculitis, two with mixed cryoglobulinaemia, and five with infectious mononucleosis. Felty's syndrome was defined by the association of classical RA, splenomegaly, and white blood counts (VBC) ranging between 500 and 2800 per mm3. In addition, sera from twenty-nine patients with systemic lupus erythematosis (SLE) were examined for the presence of granulocyte-reactive antinuclear antibodies (ANA). All the Felty patients had VBC counts under 2800 per mm3 and PMN counts under 2000 per mm3. A comparison of the Felty and RA patients is shown in Table 1. All patients in both groups were rheumatoid-factor positive and the geometric mean sensitized sheep-cell-agglutination titre was the same in both groups. The mean erythrocytesedimentation rate (ESR) was 57 mm for the RA group and 64 mm for the Felty patients. The mean WBC count for the RA group was 7500 per mm3 and for the Felty group 2600 per mm3. The Felty patients had a higher incidence of ANA and LE cells than the RA patients. Separation of white blood cells for direct immunofluorescent staining. The technique utilized in the study of intracellular inclusions has been described in a previous study (Hurd, LoSpalluto & Ziff, 1970). Fresh, heparinized blood was allowed to stand for 60-90 min, the supernatant buffy coat cells removed, centrifuged at 170 g, and washed three times in 0.900 normal saline. The buffy-coat cells were then resuspended in 0-9% saline and the concentration adjusted to 4 x 106 cells in 1-5 ml saline. Using two to three drops per slide, the cells were then centrifuged in a Shandon cytocentrifuge (Shandon Instruments, Sweickley, Pa.) onto glass slides precleaned in a solution consisting of equal parts of 95% ethanol and 0-1 N HCI for staining. In initial studies, sedimentation of the Felty patient cells was performed at room temperature. In subsequent experiments, sedimentation was performed at both 250C and 370C. Sedimentation of the cells usually required about 1 hr. Immunofluorescent staining of normal leucocytes incubated with patient sera. 4 x 106 washed normal peripheral white blood cells obtained from healthy donors were suspended in 1-0 ml of fresh patient serum and incubated for 90 min at 370C with gentle shaking. The cells were then centrifuged, washed three times in 0 9% saline and resuspended in 1-5 ml of saline. Slides of this suspension were then prepared for immunofluorescent staining by centrifugation in Shandon cytocentrifuge. For comparison, in the majority of cases, the patient's serum was also incubated with 4 x 106 of his own cells and prepared for staining as described above. Thus both normal cells and patient cells were incubated in patient serum. This method will be referred to below as the indirect-staining method. In other experiments, patient cells were isolated as described and stained directly by the immunofluorescent technique. This method will be referred to as the direct-staining method. Slides of either patient cells stained directly following separation (direct method) or of normal cells or patient cells incubated with patient serum (indirect method) were stained with fluorescein-conjugated goat antisera directed against human IgG, IgM, IgA, and B1C (Meloy Laboratories, Springfield, Virginia). The antibodies to the immunoglobulins were shown to be monospecific by immunodiffusion against purified immunoglobulins. Slides were examined in a Leitz-Ortholux microscope equipped with epi-illumination according to Ploem (1971) using an HBO 100 ultra high-pressure mercury lamp with K490 and K5 10 fluorescence filters. Cells examined for the presence of cytoplasmic inclusions were graded as follows: 0 = no significant staining of neutrophils; trace = tiny to small inclusions in over 50% of cells; 2+ = small to moderate-sized inclusions in less than 50% of cells, occasional cells containing large inclusions; 3 + = large inclusions seen in less than 500% of cells with the majority of cells containing smaller inclusions; 4+ = large inclusions in over 50% of cells. Control preparations in the direct method consisted of normal cells not previously incubated in serum. Control preparations in the indirect method consisted of normal cells incubated in the donor's own serum. TABLE 1. Comparison of classical RA and Felty's syndrome
patients
RA Latex per cent positive
SSCA titre* ESR (mm) WBC (mean) ANA per cent positive LE Cell per cent positive *
Classical RA n= 15
Felty's syndrome
100 8 57 7500 47 14
100 8 64 2600 62 46
Log2 geometric mean titre.
n= 15
Phagocytosis of immune complexes
415
Granulocyte-reactive anti-nuclear antibodies (ANA). The technique of Faber & Elling (1966) was used to examine these antibodies. Whole blood smears from normal persons were used as substrate. These were allowed to air dry and were then fixed in 95%4 ethanol for 45 min at room temperature. The patient sera were layered onto the blood smear, incubated in a moist chamber for 1 hr and washed with phosphate-buffered saline for 30 min. The slide was then stained with fluorescein-conjugated anti-IgG, IgM, IgA and B1C in a moist chamber for 30 min, and then washed and read. Ten-fold serum dilutions were made and the end point determined by reading as positive the last dilution which produced visible staining. In order to better determine the specificity of the ANA a Ficoll-Hypaque density gradient (Boyum, 1968) was used to separate populations of PMN and mononuclear cells. In this procedure 20-30 ml of peripheral blood were obtained from each subject in a heparinized syringe. The freshly drawn blood was added to an equal volume of sterile saline and mixed. 1 vol. of the blood-saline mixture was layered in a sterile centrifuge tube over 1-5 vol. of a Ficoll-Hypaque mixture made up of 10 parts of Hypaque and 24 parts of Ficoll. The mixture was then centrifuged at 400 g at room temperature for 40 min. The supernatant was discarded and the mononuclear cell band transferred to another tube in siliconized pipettes. The cells were washed three times with normal saline at room temperature and resuspended at a concentration of 3-4 x 106 cells per ml. Aliquots were then removed and cytocentrifuge preparations made for immunofluorescent staining as described above. Using the same gradients, the red-cell-PMN layer was separated and resuspended in sufficient saline to bring the total volume to the original blood volume. 6% Dextran 70, 2 5 ml were added to each 10 ml of the blood-saline mixture and the mixture allowed to stand at room temperature for 2 hr. During this time the red cells settled and the PMN remained in suspension. The PMN layer was removed, the cells were washed in Hanks' buffer and cytocentrifuge preparations prepared for staining as previously described. By this technique, the cells obtained were 98%4 PMN cells. In addition to the PMN-ANA, routine ANA tests were performed using KB-cell lines as substrate (Virgo, Electro-Nucleonics Laboratories, Bethesda, Maryland). Ten-fold serum dilutions were made to measure the ANA titre. Tests for rheumatoid factor. These tests were performed on sera by the sensitized sheep-cell-agglutination (SSCA) test, sensitizing with one-fourth the basic agglutinating titre of amboceptor. Statistical calculations. The Mann-Whitney U test (Siegel, 1956) was used for computer programming to determine significant differences between inclusion scores in Felty's syndrome and rheumatoid arthritis groups.
RESULTS Control preparations Using the direct immunofluorescent-staining method in which leucocytes were stained without prior incubation with serum, normal leucocytes showed no staining. In the indirect method, in which normal leucocytes were incubated with serum, inclusions graded as 2+ were observed in the case of two of ten normal sera examined. In the case of one serum they stained with anti-IgG only and the case of the
large intracytoplasmic vacuoles. (Magnification x 420.)
R.
416 other with anti-131C
2+ inclusions,
only.
only
Hurd,
The remainder
were
and 4+ inclusions
3
M. Andreis & M.
entirely negative.
were
considered
to
Zi/f
Because of the presence of these isolated be
significant.
Direct method In four of the fifteen patients with
inclusions
were seen in
FIG. 2.
gated
Peripheral
anti-human
Felty's syndrome,
the majority of the
large (3 +
very
circulating granulocytes
to
4±) IgG-
when these
were
and
IgM-staining
stained
immediately
blood PMN from a patient with Felty's syndrome stained directly with fluoresccin-conjuIgG demonstrating very large intracytoplasmic inclusions. (Magnification x 420.)
TABLE 2. Immunofluorescent staining of inclusions in normal polymorphonuclear cells following incubation in serum of patients with Felty's syndrome. Indirect method
Inclusions
Inclusion*
Patients
IgG
1gM
IgA
B31C
score
IlMW 3/05/73 5/08/73
4+
4+ 4+
4-1
2 VP 3 DM 9/27/73
414+I 3± 4-
4/03/74
Tr
0
16 16 8 16 It 5-5 0 7 14 8-5 8 I11 7 7 0 12 9 3 11
4-4 3+ 4-4
4 ES
4+
1+
50OE 1/24/74 6/10/75
0
0
2+
24-
6 A~a 7 LCu 8 DB 9 LWy
4-, 4+
41 2+
3+ 3+ 3+
2-+
I1OCGr 9/06/74 12/11/74
li1jEng 1 2JR 12 JS 14 ML 15 DA
2+
2}-
14
0 4+ 3+
0 3 3, Tr 2+
2+ 3 -4
2+
4-0
4± +
4-4
0 0 0
Tr Tr U 2-1
1+ 2+
441
Tr 2 3121-
24 lj13-2'
210
2j
2 0 Tr 3+
3-1 3-, 0 3-1
0
* Inclusion score deriv ed by addition of numerical values for indiv idual Igs and BIC. Trace staining (Tr) was assigned a value of 0-5.
t Patient
on
gold therapy.
Phagocytosis of immune complexes
417
following 1 hr of sedimentation of the blood at room temperature (R.T.) (Figs 1 and 2). C3 stained as B1C was also identified in the large inclusions of two of the four positive patients. Twenty-one patients, the majority of whom had high sensitized sheep-cell agglutination (SSCA) titres of rheumatoid factor (average titre 1:289, range 1: 8-1: 1792), failed to show significant inclusions by the direct-staining method for IgG. Two showed 2+ inclusions staining for IgM, and one of twenty tested showed minimal staining for C3. By the direct method inclusions were also not seen in two patients
FIG. 3. PMN from normal donor after incubation in Felty serum (indirect method) and staining with fluorescein-conjugated anti-human IgG. Very large intracytoplasmic inclusions are present. (Magnification x 420.)
TABLE 3. Immunofluorescent staining of normal polymorphonuclear cells following incubation in serum of patients with classical rheumatoid arthritis. Indirect method
Inclusions Inclusion* Patients 1 JG 2 FV 3 BV 4 CF 5 LP 6 OS 7 LW 8 RP 9 AW 10 HL 3/30/74
7/16/74 11 SJ 12 AM 9/19/73
5/22/74 13 HR 14 OB 15 RS 7/15/74 2/11/75
IgG
IgM
IgA
BIC
score
0
Tr Tr 0 0 0 0 0 0
0 0 0 Tr 0 0
Tr Tr 0 Tr 0
1 3 05 15 3 1-5 8 1 4 4-5 1-5 05 2 3 4
2+ Tr Tr
3+ Tr
3+ Tr
2+ 3+ 1+
1+
Tr Tr
1+ 0 0 0 0 0 0 0
2+
2+
Tr 2+ 2+
2+
2+ 0 0 Tr 0 0 0 0 0 Tr 0 Tr
1+ 3+ Tr
1+ 0 Tr 0 0
1+ 2+ Tr
1+ 3+
1V5 1-5 7-5
* Inclusion score derived by addition of numerical values for individual Igs and
BIC. A trace (Tr) was assigned a value of 0 5.
E. R. Hurd, M. Andreis L M. Ziff
418
with rheumatoid vasculitis and two with Sj6gren's syndrome. Significant (3+) inclusions staining for both IgG and IgM were observed in one of five patients with infectious mononucleosis. One patient with mixed cryoglobulinaemia was negative and one had 4+ inclusions which stained for IgG, 3+ for IgA and 2+ for IgM. Indirect method By the indirect technique in which normal leucocytes were incubated with patient serum (Tables 2 and 4), twelve of the fifteen patients with Felty's syndrome (80%) had significant (3+ to 4+) IgGstaining inclusions; six (40%) had significant IgM-staining inclusions; three (20%) had significant IgAand seven (47%) had significant B1C-staining inclusions. An example of positively-staining inclusions by the indirect method is shown in Fig. 3. In the other groups (Tables 3 and 4) three of fifteen patients (20%) with classical RA, one of two with rheumatoid vasculitis, two of three with Sjogren's syndrome, and one of two with mixed cryoglobulinaemia showed significant inclusions which stained for IgG. In addition, two of the fifteen RA patients (13%) and one of the two patients with mixed cryoglobulinaemia developed inclusions which stained for B1C. One of the Sjogren's patients developed inclusions staining for IgM. No significant staining was seen in the case of the normal controls. An inclusion score was calculated by addition of the numerical values, e.g. 1+, 2+, etc. for the individual Igs and BjC. A trace of staining was assigned a value of 0 5. Individual scores are shown in Tables 2 and 3 for the Felty and RA patients and the mean scores for these two groups are shown in Table 5. The Felty group had an average inclusion score of 8-4 (range 0-16) as compared with the RA group with a score of 2-7 (0.5-8). This difference was statistically significant with a P value of
Phagocytosis of immune complexes by polymorphonuclear leucocytes in patients with Felty's syndrome* E. R. HURD,t M. ANDRE I S & M. ZIFF§ Dallas Veterans Administration Hospital, Dallas, and The University of Texas Health Science Center, Dallas, Texas
(Received 29 November 1976) SUMMARY
The possible role of phagocytosis of circulating immune complexes by neutrophils in the production of the neutropenia of Felty's syndrome has been investigated. Normal neutrophils phagocytosed massive inclusions from the sera from twelve of fifteen patients with Felty's syndrome when incubated with these sera. Such inclusions were phagocytosed from only three of fifteen patients with seropositive RA who did not have Felty's syndrome. Normal neutrophils were more effective than patient neutrophils with regard to phagocytosis of inclusions from the patients' serum suggesting a defect in phagocytic function of Felty's neutrophils. The titre of granulocyte-reactive antinuclear antibodies did not appear to be related to the degree of neutropenia. The data suggest that phagocytosis of circulating immune complexes by neutrophils may interfere with the function of these cells in combating infection and also render them susceptible to removal from the circulation thus leading to the development of neutropenia.
INTRODUCTION Felty's syndrome consists of the triad of rheumatoid arthritis, leukopenia and splenomegaly (Felty, 1924). Patients with this syndrome may have frequent infections (De Gruchy & Langley, 1961; Barnes, Turnbull & Vernon-Roberts, 1971; Denko & Zumpft, 1962; Ruderman, Miller & Pinals, 1968; Hahn, Mayne & Kiely, 1963). They usually have high titres of rheumatoid factor and frequently, positive tests for antinuclear antibodies and LE cells (DeGruchy & Langley, 1961; Barnes et al., 1971; Denko & Zumpft, 1962; Ruderman et al., 1968; Hahn et al., 1963). The patients tend to have severe, though frequently inactive arthritis, subcutaneous nodules, lymphadenopathy, anemia, thrombocytopenia, manifestations of vasculitis such as chronic leg ulcers and peripheral neuropathy and an increased incidence of Sjogren's syndrome (DeGruchy & Langley, 1961; Barnes et al., 1971; Denko & Zumpft, 1962; Ruderman et al., 1968; Hahn et al., 1963). The leukopenia is due primarly to a neutropenia and is associated with an increase in myeloid activity in the bone marrow (DeGruchy & Langley, 1961). The etiology of the leukopenia has been obscure, although proposed mechanisms have included neutrophil-specific antinuclear antibodies (Faber & Elling, 1966), margination of the neutrophils (Vincent, Levi & Macqueen, 1974) and impaired release of neutrophils from the bone marrow (Brubaker, Johnson & Morris, 1974). The present study presents data which suggests another possible mechanism for the neutropenia, i.e. phagocytosis of circulating immune complexes by the circulating granulocytes. Presented in part before the 4th Pan American Congress of Rheumatology, Toronto, Ontario, Canada, June, 1974. t Clinical Scholar, Arthritis Foundation and previously, Clinical Investigator, Dallas V.A. Hospital, and Associate Professor of Internal Medicine, The University of Texas Health Science Center, Dallas. t Assistant Professor of Internal Medicine, The University of Texas Health Science Center, Dallas; Postdoctoral Fellow, The Arthritis Foundation. § Professor of Internal Medicine, The University of Texas Health Science Center, Dallas; Recipient, Research Career Award, US Public Health Service. Correspondence: Dr Eric R. Hurd, Department of Internal Medicine, The University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, U.S.A. *
413
414
E. R. Hurd, M. Andreis & M. Ziff MATERIALS AND METHODS
Patient and control population. Studies were performed on the blood taken from ten normal persons, fifteen with Felty's syndrome, fifteen with classical rheumatoid arthritis (RA) as defined by the ARA diagnostic criteria (Ropes et al., 1959; Cohen et al., 1971), but without evidence of splenomegaly or leukopenia, three with Sjogren's syndrome, two with rheumatoid vasculitis, two with mixed cryoglobulinaemia, and five with infectious mononucleosis. Felty's syndrome was defined by the association of classical RA, splenomegaly, and white blood counts (VBC) ranging between 500 and 2800 per mm3. In addition, sera from twenty-nine patients with systemic lupus erythematosis (SLE) were examined for the presence of granulocyte-reactive antinuclear antibodies (ANA). All the Felty patients had VBC counts under 2800 per mm3 and PMN counts under 2000 per mm3. A comparison of the Felty and RA patients is shown in Table 1. All patients in both groups were rheumatoid-factor positive and the geometric mean sensitized sheep-cell-agglutination titre was the same in both groups. The mean erythrocytesedimentation rate (ESR) was 57 mm for the RA group and 64 mm for the Felty patients. The mean WBC count for the RA group was 7500 per mm3 and for the Felty group 2600 per mm3. The Felty patients had a higher incidence of ANA and LE cells than the RA patients. Separation of white blood cells for direct immunofluorescent staining. The technique utilized in the study of intracellular inclusions has been described in a previous study (Hurd, LoSpalluto & Ziff, 1970). Fresh, heparinized blood was allowed to stand for 60-90 min, the supernatant buffy coat cells removed, centrifuged at 170 g, and washed three times in 0.900 normal saline. The buffy-coat cells were then resuspended in 0-9% saline and the concentration adjusted to 4 x 106 cells in 1-5 ml saline. Using two to three drops per slide, the cells were then centrifuged in a Shandon cytocentrifuge (Shandon Instruments, Sweickley, Pa.) onto glass slides precleaned in a solution consisting of equal parts of 95% ethanol and 0-1 N HCI for staining. In initial studies, sedimentation of the Felty patient cells was performed at room temperature. In subsequent experiments, sedimentation was performed at both 250C and 370C. Sedimentation of the cells usually required about 1 hr. Immunofluorescent staining of normal leucocytes incubated with patient sera. 4 x 106 washed normal peripheral white blood cells obtained from healthy donors were suspended in 1-0 ml of fresh patient serum and incubated for 90 min at 370C with gentle shaking. The cells were then centrifuged, washed three times in 0 9% saline and resuspended in 1-5 ml of saline. Slides of this suspension were then prepared for immunofluorescent staining by centrifugation in Shandon cytocentrifuge. For comparison, in the majority of cases, the patient's serum was also incubated with 4 x 106 of his own cells and prepared for staining as described above. Thus both normal cells and patient cells were incubated in patient serum. This method will be referred to below as the indirect-staining method. In other experiments, patient cells were isolated as described and stained directly by the immunofluorescent technique. This method will be referred to as the direct-staining method. Slides of either patient cells stained directly following separation (direct method) or of normal cells or patient cells incubated with patient serum (indirect method) were stained with fluorescein-conjugated goat antisera directed against human IgG, IgM, IgA, and B1C (Meloy Laboratories, Springfield, Virginia). The antibodies to the immunoglobulins were shown to be monospecific by immunodiffusion against purified immunoglobulins. Slides were examined in a Leitz-Ortholux microscope equipped with epi-illumination according to Ploem (1971) using an HBO 100 ultra high-pressure mercury lamp with K490 and K5 10 fluorescence filters. Cells examined for the presence of cytoplasmic inclusions were graded as follows: 0 = no significant staining of neutrophils; trace = tiny to small inclusions in over 50% of cells; 2+ = small to moderate-sized inclusions in less than 50% of cells, occasional cells containing large inclusions; 3 + = large inclusions seen in less than 500% of cells with the majority of cells containing smaller inclusions; 4+ = large inclusions in over 50% of cells. Control preparations in the direct method consisted of normal cells not previously incubated in serum. Control preparations in the indirect method consisted of normal cells incubated in the donor's own serum. TABLE 1. Comparison of classical RA and Felty's syndrome
patients
RA Latex per cent positive
SSCA titre* ESR (mm) WBC (mean) ANA per cent positive LE Cell per cent positive *
Classical RA n= 15
Felty's syndrome
100 8 57 7500 47 14
100 8 64 2600 62 46
Log2 geometric mean titre.
n= 15
Phagocytosis of immune complexes
415
Granulocyte-reactive anti-nuclear antibodies (ANA). The technique of Faber & Elling (1966) was used to examine these antibodies. Whole blood smears from normal persons were used as substrate. These were allowed to air dry and were then fixed in 95%4 ethanol for 45 min at room temperature. The patient sera were layered onto the blood smear, incubated in a moist chamber for 1 hr and washed with phosphate-buffered saline for 30 min. The slide was then stained with fluorescein-conjugated anti-IgG, IgM, IgA and B1C in a moist chamber for 30 min, and then washed and read. Ten-fold serum dilutions were made and the end point determined by reading as positive the last dilution which produced visible staining. In order to better determine the specificity of the ANA a Ficoll-Hypaque density gradient (Boyum, 1968) was used to separate populations of PMN and mononuclear cells. In this procedure 20-30 ml of peripheral blood were obtained from each subject in a heparinized syringe. The freshly drawn blood was added to an equal volume of sterile saline and mixed. 1 vol. of the blood-saline mixture was layered in a sterile centrifuge tube over 1-5 vol. of a Ficoll-Hypaque mixture made up of 10 parts of Hypaque and 24 parts of Ficoll. The mixture was then centrifuged at 400 g at room temperature for 40 min. The supernatant was discarded and the mononuclear cell band transferred to another tube in siliconized pipettes. The cells were washed three times with normal saline at room temperature and resuspended at a concentration of 3-4 x 106 cells per ml. Aliquots were then removed and cytocentrifuge preparations made for immunofluorescent staining as described above. Using the same gradients, the red-cell-PMN layer was separated and resuspended in sufficient saline to bring the total volume to the original blood volume. 6% Dextran 70, 2 5 ml were added to each 10 ml of the blood-saline mixture and the mixture allowed to stand at room temperature for 2 hr. During this time the red cells settled and the PMN remained in suspension. The PMN layer was removed, the cells were washed in Hanks' buffer and cytocentrifuge preparations prepared for staining as previously described. By this technique, the cells obtained were 98%4 PMN cells. In addition to the PMN-ANA, routine ANA tests were performed using KB-cell lines as substrate (Virgo, Electro-Nucleonics Laboratories, Bethesda, Maryland). Ten-fold serum dilutions were made to measure the ANA titre. Tests for rheumatoid factor. These tests were performed on sera by the sensitized sheep-cell-agglutination (SSCA) test, sensitizing with one-fourth the basic agglutinating titre of amboceptor. Statistical calculations. The Mann-Whitney U test (Siegel, 1956) was used for computer programming to determine significant differences between inclusion scores in Felty's syndrome and rheumatoid arthritis groups.
RESULTS Control preparations Using the direct immunofluorescent-staining method in which leucocytes were stained without prior incubation with serum, normal leucocytes showed no staining. In the indirect method, in which normal leucocytes were incubated with serum, inclusions graded as 2+ were observed in the case of two of ten normal sera examined. In the case of one serum they stained with anti-IgG only and the case of the
large intracytoplasmic vacuoles. (Magnification x 420.)
R.
416 other with anti-131C
2+ inclusions,
only.
only
Hurd,
The remainder
were
and 4+ inclusions
3
M. Andreis & M.
entirely negative.
were
considered
to
Zi/f
Because of the presence of these isolated be
significant.
Direct method In four of the fifteen patients with
inclusions
were seen in
FIG. 2.
gated
Peripheral
anti-human
Felty's syndrome,
the majority of the
large (3 +
very
circulating granulocytes
to
4±) IgG-
when these
were
and
IgM-staining
stained
immediately
blood PMN from a patient with Felty's syndrome stained directly with fluoresccin-conjuIgG demonstrating very large intracytoplasmic inclusions. (Magnification x 420.)
TABLE 2. Immunofluorescent staining of inclusions in normal polymorphonuclear cells following incubation in serum of patients with Felty's syndrome. Indirect method
Inclusions
Inclusion*
Patients
IgG
1gM
IgA
B31C
score
IlMW 3/05/73 5/08/73
4+
4+ 4+
4-1
2 VP 3 DM 9/27/73
414+I 3± 4-
4/03/74
Tr
0
16 16 8 16 It 5-5 0 7 14 8-5 8 I11 7 7 0 12 9 3 11
4-4 3+ 4-4
4 ES
4+
1+
50OE 1/24/74 6/10/75
0
0
2+
24-
6 A~a 7 LCu 8 DB 9 LWy
4-, 4+
41 2+
3+ 3+ 3+
2-+
I1OCGr 9/06/74 12/11/74
li1jEng 1 2JR 12 JS 14 ML 15 DA
2+
2}-
14
0 4+ 3+
0 3 3, Tr 2+
2+ 3 -4
2+
4-0
4± +
4-4
0 0 0
Tr Tr U 2-1
1+ 2+
441
Tr 2 3121-
24 lj13-2'
210
2j
2 0 Tr 3+
3-1 3-, 0 3-1
0
* Inclusion score deriv ed by addition of numerical values for indiv idual Igs and BIC. Trace staining (Tr) was assigned a value of 0-5.
t Patient
on
gold therapy.
Phagocytosis of immune complexes
417
following 1 hr of sedimentation of the blood at room temperature (R.T.) (Figs 1 and 2). C3 stained as B1C was also identified in the large inclusions of two of the four positive patients. Twenty-one patients, the majority of whom had high sensitized sheep-cell agglutination (SSCA) titres of rheumatoid factor (average titre 1:289, range 1: 8-1: 1792), failed to show significant inclusions by the direct-staining method for IgG. Two showed 2+ inclusions staining for IgM, and one of twenty tested showed minimal staining for C3. By the direct method inclusions were also not seen in two patients
FIG. 3. PMN from normal donor after incubation in Felty serum (indirect method) and staining with fluorescein-conjugated anti-human IgG. Very large intracytoplasmic inclusions are present. (Magnification x 420.)
TABLE 3. Immunofluorescent staining of normal polymorphonuclear cells following incubation in serum of patients with classical rheumatoid arthritis. Indirect method
Inclusions Inclusion* Patients 1 JG 2 FV 3 BV 4 CF 5 LP 6 OS 7 LW 8 RP 9 AW 10 HL 3/30/74
7/16/74 11 SJ 12 AM 9/19/73
5/22/74 13 HR 14 OB 15 RS 7/15/74 2/11/75
IgG
IgM
IgA
BIC
score
0
Tr Tr 0 0 0 0 0 0
0 0 0 Tr 0 0
Tr Tr 0 Tr 0
1 3 05 15 3 1-5 8 1 4 4-5 1-5 05 2 3 4
2+ Tr Tr
3+ Tr
3+ Tr
2+ 3+ 1+
1+
Tr Tr
1+ 0 0 0 0 0 0 0
2+
2+
Tr 2+ 2+
2+
2+ 0 0 Tr 0 0 0 0 0 Tr 0 Tr
1+ 3+ Tr
1+ 0 Tr 0 0
1+ 2+ Tr
1+ 3+
1V5 1-5 7-5
* Inclusion score derived by addition of numerical values for individual Igs and
BIC. A trace (Tr) was assigned a value of 0 5.
E. R. Hurd, M. Andreis L M. Ziff
418
with rheumatoid vasculitis and two with Sj6gren's syndrome. Significant (3+) inclusions staining for both IgG and IgM were observed in one of five patients with infectious mononucleosis. One patient with mixed cryoglobulinaemia was negative and one had 4+ inclusions which stained for IgG, 3+ for IgA and 2+ for IgM. Indirect method By the indirect technique in which normal leucocytes were incubated with patient serum (Tables 2 and 4), twelve of the fifteen patients with Felty's syndrome (80%) had significant (3+ to 4+) IgGstaining inclusions; six (40%) had significant IgM-staining inclusions; three (20%) had significant IgAand seven (47%) had significant B1C-staining inclusions. An example of positively-staining inclusions by the indirect method is shown in Fig. 3. In the other groups (Tables 3 and 4) three of fifteen patients (20%) with classical RA, one of two with rheumatoid vasculitis, two of three with Sjogren's syndrome, and one of two with mixed cryoglobulinaemia showed significant inclusions which stained for IgG. In addition, two of the fifteen RA patients (13%) and one of the two patients with mixed cryoglobulinaemia developed inclusions which stained for B1C. One of the Sjogren's patients developed inclusions staining for IgM. No significant staining was seen in the case of the normal controls. An inclusion score was calculated by addition of the numerical values, e.g. 1+, 2+, etc. for the individual Igs and BjC. A trace of staining was assigned a value of 0 5. Individual scores are shown in Tables 2 and 3 for the Felty and RA patients and the mean scores for these two groups are shown in Table 5. The Felty group had an average inclusion score of 8-4 (range 0-16) as compared with the RA group with a score of 2-7 (0.5-8). This difference was statistically significant with a P value of
