Scand J H a e m a t o l ( l 9 7 5 ) 15, 27-34
An Inhibitor of Chemotaxis and Phagocytosis in Reticulum Cell Sarcoma T. Rumu, M.D.,P. Rumu, M.D., P. VUOPIO, M.D.,K. FRANSSILA, M.D. & E. LINDER, M.D. First Department (Chief, P . I . Halonen) and Third Department (Chief, E . A . Nikkila) of Medicine, Department of Serology and Bacteriology (Acting Chief, T . U . Kosunen) and Second Department of Pathology (Chief, H . Teir), University o f Helsinki, Helsinki, Finland
In a study of neutrophil functions in haematological disorders using in vitro techniques, a heat-stable inhibitor of chemotaxis and phagocytosis was demonstrated in the plasma and serum of a 64-year-old woman with reticulum cell sarcoma. In partial purification by chromatography, the inhibitory activity could not be separated from IgG. Clinically the patient did not exhibit abnormal susceptibility to infections. K e y words: chemotaxis - phagocytosis - inhibitor - reticulosarcoma
Accepted for publication May 16, 1975 Correspondence to: Dr. P. Ruutu, Department of Serology and Bacteriology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki 29, Finland
During the last few years a wide spectrum of defects in the phagacytic process have been described, many of them associated with increased susceptibility to infections. Chemotaxis, phagocytosis, or intracellular killing of microorganisms may be affected (Stossel 1974). Some disorders of chemotaxis are caused by inhibitors. Such inhibitors have been demonstrated in some children suffering from severe infections (Ward & Schlegel 1969, Smith et a1 1972, Soriano et a1 1973), in chronic hypommplementaemic glomerulonephritis (Gewurz et a1 1967), and in hepatic cirrhosis (DeMeo & Andersen
1972). An inhibitor of chemotaxis is also present in low concentration in normal sera, and it is obviously one component of the homeostatic control system of chemotaxis (Berenberg & Ward 1973). In Hodgkin’s disease (Ward & Berenberg 1974) and in Bruton-type agammaglobulinaemia (Ward 1972) abnormal chemotaxis-inhibiting activity has been observed in the serum; this activity was probably caused by the physiological inhibitor in abnormally high concentration. There are few reports on serum inhibitors of phagocytosis. Penny & Galton (1966) demonstrated defective neutrophil phagocytosis in myelomatosis and macro-
28
T. RUUTU, P. RUUTU, P.VUOPI0, K. FRANSSILA & E. LINDER
globulinaemia, caused by inhibitory activity in plasma. In this paper we describe an inhibitor of chemotaxis and phagocytosis in a woman with reticulum cell sarcoma. CASE REPORT A 64-year-old housewife was referred to the University Central Hospital, Helsinki, in May 1970 because of lymphadenopathy and thrombocytopenia of several months’ duration. Enlarged lymph nodes, up t o 2 cm in diameter, were found in the neck, supraclavicular fossae, axillae and groins. The liver and the spleen were palpable. Slight normochromic anaemia and a normal leucocyte count with slight neutropenia were noted. The number of platelets was normal. In the bone marrow 10-20 % of the cells were atypical large blast cells, and the same cell type was also found in spleen aspirate. A lymph node biopsy specimen showed nonspecific follicular hyperplasia. Lymphography revealed enlargement of paraaortic lymph nodes. The patient was closely followed for the next 3 years. In autumn 1970 bleedings occurred and thrombocytopenia was observed. The life span of SICr-labelled platelets was one-third of the normal and splenectomy was performed. The spleen weighed 700 g. Some extramedullary haematopoiesis was seen microscopically, but nothing t o indicate malignancy. The enlarged lymph nudes at the hilus of the spleen were hyperplastic. The constant histological finding on repeated biopsies of the persistently enlarged lymph nodes was nonspecific hyperplasia. After the splenectomy the number of platelets was constantly normal. There was usually slight leucocytosis with a normal differential count. In bone marrow aspirates up t o 15 % atypical blast cells were seen at times. Bone marrow chromosome analysis was normal. The leucocyte alkaline phosphatase score was constantly low, 2 to 20. There was no lysozyme in the urine. The ESR (Westergren 1 h) varied between 6 and 28 mm. The serum gammaglobulins were increased, 17-25 g/l, and IgG, JgA and IgM were all polyclonally elevated. N o Bence Jones protein was found. The latex test and antinuclear antibodies were negative in the beginning, later positive. No LE-cells were seen. Tests for sarcoidosis, tuberculosis, mononucleosis, brucel-
losis, tularaemia, leptospirosis, yersinia infection, toxoplasmosis and listeriosis as well as screening for viral antibodies gave negative or normal results. The concentration of C‘3 was 0.76 g/], that of C’4 0.51 g/l. The lymphocyte response to phytohaemagglutinin was diminished. In lymphography the paraaortic lymph nodes were constantly enlarged. N o mediastinal lymph node enlargement was seen. Epigastric pains and loss of weight appeared in summer 1973. Gastroscopy revealed a cardiac tumour mass. Histologically the tumour was difficult to classify, but the mmt probable diagnosis was reticulum cell sarcoma. At laparotomy the tumour was found to extend as far as the hepatic flexure and far into the retroperitoneal space. No active surgery was performed. The patient died in August 1973, two days after the operation. At autopsy a flat tumour was found to encircle the distal part of the eosophagus and the cardia. Similar tumour growth was also found in the middle of the small intestine, the wall of which was perforated resulting in bacterial peritonitis. Histologically the tumour was a reticulum cell sarcoma. Microscopic examination of the enlarged paraaortic lymph nodes revealed nonspecific hyperplasia. No treatment, except occasional symptomatic medication, was given during the course of the disease because of the lack of diagnostic histological findings in the lymph node specimens. METHODS Phago cy tosis
This method has been described in detail elsewhere (Ruutu 1972). From fresh hepannized blood most of the erythrocytes were removed by Boyum’s method (1968). Cells separated from leucocyte-rich plasma were washed once with Hanks’ solution containing 1 % of bovine serum albumin and suspended in plasma, serum, or Hanks’ solution containing plasma or serum, as indicated. For the assay, Staphylococcus epidermidis, killed with formalin and washed with saline, was added to the leucocyte suspension in the relation 5 bacteria t o one neutrophil. The tubes were incubated at 37O C and mixed at intervals. After 6 min smears for microscopy were made and stained with Wright’s stain. Phagocytic index,
INHIBITOR OF CHEMOTAXIS AND PHAGOCYTOSIS the average number of bacteria phagocytosed by one neutrophil, was determined from 200 cells. Bactericidal activity of leucocytes
To measure the ability of leucocytes to kill Staphylococcus aureus, a slight modification (Ruutu 1972) of the method of Holmes et a1 (1966) was used
29
activity and assayed for IgG content and albumin content by radial immunodiffusion. For technical reasons fractions of patient and normal sera were tested on successive days with simultaneo’us controls.
RESULTS
Phagocytosis (Table I) Chemotaxis
A modification of the Boyden technique (1962) was used. The chamber was divided into an upper and a lower compartment by a membrane with an average pore diameter of 3.0 ,um (Millipore MF membranes). The chemotactic stimulus was prepared by incubating serum with zymosan (1 mg zymosan par 1 ml serum) for 1 h at 37O C, and diluted with Hank’s solution as indicated. Fresh frozen pooled serum from healthy donors was used as normal serum. The cells were suspended in Hanks’ solution containing 1 % of b e vine serum albumin at a concentration of 2 x lo6 granulocytes per ml. The chambers, containing 1 ml of chemotactic stimulus in the lower compartment and 1 ml of cell suspension in the upper compartment, were incubated at 37OC for 3 h. The filters were then removed, fixed in ethanol, stained with haematoxylin, dried, and cleared in xylene. The number of cells that had migrated through the filter t o the lower surface was counted at a magnification of 400 x in 10 random fileds. Chemotactic activity was expressed as the average number of cells per highpower field. All tests were done in triplicate. Serum fractionation
Partial purification of the factor@) responsible for the inhibition of chemotaxis and phagocytosis in the serum was achieved by column chromatography on Sephadex G-150 (Pharmacia) in 0.1 M Tris-HC1 buffer at pH 8.3. Pooled fractions to be tested for inhibiting activity were passed through a bacterial filter with pore size 0.22 ,U (Falcon plastics) and dialyzed against Hanks’ salt solution. The fractions were then concentrated by negative pressure dialysis t o a volume of 5 x the serum volume subjected t o fractionation. The concentrated fractions were tested for inhibitor
Phagocytosis by the patient neutrophils was deficient when the incubation medium contained patient plasma and normal in the presence of normal plasma. The same was true for cells from healthy controls: when plasma from the patient was used, phagocytosis was impaired. It is obvious that the inhibition of phagocytosis was plasmaassociated and that the cells were normal in their function. Serum behaved like plasma. When both patient and normal plasma were used in the phagocytosis assay, phagocytosis was impaired. This indicates that the defect of phagocytosis was caused by an inhibitor. If plasma lacking some factor necessary for phagocytosis is added to a system containing normal plasma, this may be expected to have no influence on phagocytosis or to increase it, since an increase of the plasma concentration enhances phagocytosis. The inhibition of phagocytosis was roughly the same in relation to the control whether 80 or 10 % of plasma was used in the incubation medium. Heating of the serum for 30 min at 5 6 O C did not affect the inhibition of phagocytosis. In our standard phagocytosis assay, mixing is not continuous in order to avoid clumping of cells. To exclude the possibility that this type of mixing would allow chemotaxis to influence the results of the phagocytosis tests, experiments were also made
T. RUUTU, P. RUUTU, P.VUOPl0, K. FRANSSILA & E. LINDER
30
Cells Plasma (or serum) concentration
I
I
Comment
PC PC NC NC
PP NP PP NP
80% 80% 80% 80%
PC PC PC PC NC NC NC NC
NP NP NP PP NP NP NP PP
20% 10% 1 0 % + PP 1 0 % 10% 20% 10% 10% + PP 1 0 % 10%
NC NC
PS 1 0 % NS 1 0 %
Similar defect in serum
NC NC NC NC
NP PP NP PP
80% 80% 10% 10%
Roughly unchanged inhibition with different plasma concentrations
NC NC NC NC
NS NS NS NS
80% 10 % + NS 70 % 30 min 5 6 O C l O % + P S 70% 10 % + PS 70 % 30 min 56O C
The inhibitor is heat-stable
The defect is plasma-associated
The defect is caused by an inhibitor
PI = phagocytic index, phagocytosed bacteria per one neutrophil. NC = normal cells, PC = patient cells, NP = normal plasma, PP = patient plasma, NS = normal serum, PS = patient serum.
with continuous mixing on an inclined wheel (not shown in the table). The inhibition of phagocytosis by patient plasma was at least as strong as in the standard assay. The inhibition of phagocytosis was demonstrated for the first time one and half years before the patient died, and several times thereafter. Bactericidal activity
After ingestion in the presence of normal serum, the ability of the patient’s leucocytes to kill Staphylococcus aureus was normal. Chemotaxis
Methods for the study of chemotaxis were
not available while the patient was alive. Therefore, deep-frozen (- 40° C) patient serum was used in the following investigations, which include no tests with patient cells. Table I1 shows a representative chemotaxis experiment. Patient serum in the cell compartment impaired chemotaxis. When bolth normal and patient serum were added, chemotaxis was also reduced. When patient serum was used as the source of chemotactic factors in the lower compartment, chemotaxis was highly deficient. Chemotaxis was also impaired when both normal and patient serum were used in the lower chamber. These experiments show that defective chemotaxis in the presence of patient se-
31
INHIBITOR OF CHEMOTAXIS AND PHAGOCYTOSIS TABLE I1 Chemotaxis (representative experiment) Cells Per field
Lower compartment
Cell compartment
NC, 10 % NS NC, 10 % NS NC, 10 % PS NC, 5 % NS + 5 % PS NC, 5 % NS NC, 10 % NS NC, 10 % NS NC, 10 % NS NC, 10 % NS NC, 10 % PS heated 30 min 56O C
30 % NS, heated 30 min 56O C 30 % NS, zymosan 30 % NS, zymosan 30 % NS, zymosan 30 % NS, zymosan 3 0 % PS, zymosan 20 % NS, zymosan 20 % NS, zymosan + 30 % NS 20 % NS, zymosan + 30 % PS 30 70NS, zymosan
2 38 3 27 44 1 36 39 21 14
% of positive control
5 100
\
7: 117 J
Comment
Negative control Positive control Inhibition of chemotaxis when patient serum in cell compartment Inhibition of chemotaxis when patient serum in lower compartment Inhibition of chemotaxis after heating of serum
NC = normal cells, NS = normal serum, PS = patient serum
rum was caused in part at least by an inhibitor. Patient serum was a poor source of chemotactic activity, but for this the inhibitor may be responsible; nothing definite can thus be said about the ability of the serum to produce chemoltactic factors. Inhibition of chemotaxis was also observed after heating of the patient’s serum at 56O C for 30 min. Eflect of serum fractions on chemotaxis and phagocytosis Chromatography on Sephadex G-150, V, 120 ml, yielded inhibitory activity for both chemotaxis and phagocytosis in the second of three major protein peaks, corresponding to 134-167 ml (Figure 1). IgG activity was also demonstrated in this part of the eluate. Separation from IgG activity was attempted by dividing the eluate into smaller fractions, but also in this experiment inhibitor activity coincided with the peak of IgG activity. The higher protein concentration in the inhibitory fraction as compared with the corresponding control fraction does not ex-
plain the inhibition, at least not the inhibition of phagocytosis, since an increased concentration of serum or plasma in the assay tends to enhance phagocytosis (Table I). DISCUSSION
The present experiments show that the patient had a serum inhibitor of chemotaxis. Such an inhibitor may affect phagocytic cells (Ward & Schlegel 1969, Smith et a1 1972, Soriano et a1 1973) or chemotactic factors (Gewurz et a1 1967, DeMeo & Andersen 1972, Ward 1972, Berenberg & Ward 1973, Ward & Berenberg 1974). No definite conclusions can be drawn about the point of action of the present inhibitor. In experiments reported by others, a celldirected inhibitor was active only in the cell compartment but not in the lower compartment (Smith et a1 1972, Soriano et a1 1973), whereas a chemotactic factordirected inhibitor was active in low concentrations in the lower compartment only, in high concentrations in both compartments (Berenberg & Ward 1973). In these
32
T. RUUTU, P. RUUTU, P.VUOPI0, K. FRANSSILA & E. LINDER
experiments the effect was of roughly the same magnitude whether patient serum was added into the cell compartment or into the lower compartment. It is not surprising that an inhibitor exerts its effect independent of which compartment it is introduced into, since the filter does not hinder diffusion. It may be assumed that a chemotactic factor inhibitor in particular could be
effective even when introduced into the cell compartment, considering that the chemotactic factors must diffuse through the filter to exert their action. In the inhibition of phagocytosis, a humoral object of action seems probable, since the reduction of the ratio of plasma to cells to 1:8 did not diminish the inhibition. The cell-directed inhibitors of chemotaxis have always been
1
- 100 g
E
'ij
%.
.* 50
I
serum
I
5
Patient serum
I
04u=.-I__--.Lp
om
N
111
150
200
V
-
250
,
300
350
VI
Fraction
403
Effluentvolume ml
Figure 1. Effect of fractions of patient and normal sera on phagocytosis and chemotaxis of normal neutrophils Phagocytosis assay Chemotaxis assay Cell compartment Control Fractions Control Fractions 5 x lo6 neutrophils 5 x 106 neutrophils 2 x 106 neutrophils 2 x lo6 neutrophils 0.8 ml Hanks 0.8 ml fraction 0.9 ml Hanks + 0.5 ml Hanks + 0.1 ml normal serum 0.1 ml normal serum 0.5 % BSA 0.5 % BSA 0.1 ml bacterial 0.1 ml bacterial 0.1 ml normal serum 0.5 ml fraction suspension suspension Lower compartment 0.3 ml normal serum + zymosan 0.7 ml Hanks PI = phagocytic index
BSA = bovine serum albumin
INHIBITOR OF CHEMOTAXIS AND PHAGOCYTOSIS
associated with severe susceptibility to infections (Ward & Schlegel 1969, Smith et a1 1972, Soriano et a1 1973), but in patients with chemotactic factor inhibitor (Gewurz et a1 1967, DeMeo & Andersen 1972, Ward & Berenberg 1974) the tendency has not been so striking. The present patient was not exceptionally susceptible to infections. Therefore, inhibition of chemotactic factors seems more probable in this case. Although various agents have been shown to inhibit phagocytosis in vivo and in vitro in experimental conditions, there are very few reports on serum inhibitors of phagocytosis in patients. Penny & Galton (1966) demonstrated phagocytosis-inhibiting activity in the plasma of patients with myelomatosis and macroglobulinaemia. This activity was correlated with the concentration of M-protein. It was suggested that M-protein is adsorbed on the surface of neutrophils, and interferes with membrane potential or with the action of serum factors necessary for phagocytic ability. Sbarra et a1 (1964) described impaired phagocytic capability of leucocytes in a few patients with lymphosarcoma and chronic lymphocytic leukaemia. In some of these patients the defect could be corrected, as in the present case, by substituting normal serum for autologous serum. No effort was made to study, whether the serum-associated defect was due to the deficiency of some factor or an inhibitor. Combined defects of chemottaxis and phagocytosis have been described before (Miller & Nilsson 1970, McCall et a1 1971, Miller 1971, Steerman et a1 1971). This is not surprising considering the interaction of mechanisms (Stossel 1974). In the present case the inhibitory activities for chemotaxis and phagocytosis were found in the same serum fraction, and the same Scand J Haernatol(1975) 15
33
factor was probably responsible for both. The effects of previously described inhibitors of chemotactic factors (Gewurz et a1 1967, DeMeo & Andersen 1972, Ward & Berenberg 1974) on phagocytosis were not tested. As the inhibitory activities could not be separated from IgG by chromatography, the inhibitor may be an immunoglobulin. Further efforts to characterize the inhibitor, e.g., by ion-exchange chromatography, were prevented by a shortage of serum post mortem. The inhibitor may be an autoantibody against some humoral factor associated with chemotaxis and phagocytosis. Autoantibodies occur in malignant diseases of the lymphatic system (Seligmann et a1 1965, Sacks 1974). The mechanism of inhibition seems to be different from that in myelomatosis and macroglobulinaemia (Penny & Galton 1966). In those diseases, the inhibition is correlated, in part at least, with the concentration of M-protein, and is probably caused by this protein, as stated above. In this patient no M-protein was found. Why the disorder, which was quite severe in vitro, did not clinically affect the resistance to infection remains obscure. This investigation was supported by the Sigrid Jus6lius Foundation, Helsinki. REFERENCES Berenberg J L & Ward P A (1973) Chemotactic factor inactivator in normal human serum. J Clin Invest 52, 1200-06. Boyden S (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med 115, 453-66. Boyum A (1968) Isolation of leucocytes from human blood. Scand J Clin Lab Invest 21, Suppl. 97, 9-29. DeMeo A N & Andersen B R (1972) Defective 3
34
T. RUUTU, P. RUUTU, P.VUOPI0, K. FRANSSILA & E. LINDER
chemotaxis associated with a serum inhibitor in cirrhotic patients. N Engl J Med 286, 735-40. Gewurz H, Page A R, Pickering R J & Good R A (1967) Complement activity and inflammatory neutrophil exudation in man. Studies in patients with glomerulonephritis, essential hypocomplementemia and agammaglobulinemia. Int Arch Allergy Appl lmmunol 32, 64-90. Holmes B, Quie P G, Windhorst D B & Good R A (1966) Fatal granulomatous disease of childhood. An inborn abnormality of phagocytic function. Lancet 1, 1225-28. McCall C E, Caves J, Cooper R & DeChatelet L (1971) Functional characteristics of human toxic neutrophils. J Infect Dis 124, 68-75. Miller M E (1971) Chemotactic function in the human neonate: humoral and cellular aspects. Pediat Res 5, 487-92. Miller M E & Nilsson U R (1970) A familial deficiency of the phagocytosis-enhancing activity of serum related t o a dysfunction of the fifth component of complement (C5). N Engl J Med 282, 354-58. Penny R & Galton D A G (1966) Studies on neutrophil function. 11. Pathological aspects. Br J Haematol 12, 633-45. Ruutu T (1972) Effect of phenothiazines and related compounds on phagocytosis and bacterial killing by human neutrophilic leukocytes. Ann Med Exp Biol Fenn 50, 24-36. Sacks P V (1974) Autoimmune hematologic complications in malignant lymphoproliferative disorders. Arch Intern Med 134, 781-83. Sbarra A J, Shirley W, Selvaraj R J, Ouchi E & Rosenbaum E (1964) The role of the phagocyte
in host-parasite interactions. I. The phagocytic capabilities of leukocytes from lymphoproliferative disorders. Cancer Res 24, 1958-68. Seligmann M, Cannat A & Hamard M (1965) Studies on antinuclear antibodies. Ann N Y Acad Sci 124, 816-32. Smith C W, Hollers J C, Dupree E, Goldman A S & Lord R A (1972) A serum inhibitor of leukotaxis in a child with recurrent infections. J Lab Clin Med 79, 878-85. Soriano R B, South M A, Goldman A S & Smith C W (1973) Defect of neutrophil motility in a child with recurrent bacterial infections and disseminated cytomegalovirus infection. J Pediatr 83, 951-58. Steerman R L, Snyderman R, Leikin S L & Colten H R (1971) Intrinsic defect of the polymorphonuclear leucocyte resulting in impaired chemotaxis and phagocytosis. Clin Exp lmmunol 9, 939-46. Stossel T P (1974) Phagocytosis. N Engl J Med 290, 833-39. Ward P A (1972) Natural and synthetic inhibitors of leukotaxis. In I H Lepow & P A Ward (eds) Inflammation Mechanisms and Control, pp 30108. Academic Press, New York. Ward P A & Berenberg J L (1974) Defective regulation of inflammatory mediators in Hodgkin's disease. Supernormal levels of chemotactic-factor inactivator. N Engl J Med 290, 7680. Ward P A & Schlegel R J (1969) Impaired leucotactic responsiveness in a child with recurrent infections. Lancet 2, 344-47.
An Inhibitor of Chemotaxis and Phagocytosis in Reticulum Cell Sarcoma T. Rumu, M.D.,P. Rumu, M.D., P. VUOPIO, M.D.,K. FRANSSILA, M.D. & E. LINDER, M.D. First Department (Chief, P . I . Halonen) and Third Department (Chief, E . A . Nikkila) of Medicine, Department of Serology and Bacteriology (Acting Chief, T . U . Kosunen) and Second Department of Pathology (Chief, H . Teir), University o f Helsinki, Helsinki, Finland
In a study of neutrophil functions in haematological disorders using in vitro techniques, a heat-stable inhibitor of chemotaxis and phagocytosis was demonstrated in the plasma and serum of a 64-year-old woman with reticulum cell sarcoma. In partial purification by chromatography, the inhibitory activity could not be separated from IgG. Clinically the patient did not exhibit abnormal susceptibility to infections. K e y words: chemotaxis - phagocytosis - inhibitor - reticulosarcoma
Accepted for publication May 16, 1975 Correspondence to: Dr. P. Ruutu, Department of Serology and Bacteriology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki 29, Finland
During the last few years a wide spectrum of defects in the phagacytic process have been described, many of them associated with increased susceptibility to infections. Chemotaxis, phagocytosis, or intracellular killing of microorganisms may be affected (Stossel 1974). Some disorders of chemotaxis are caused by inhibitors. Such inhibitors have been demonstrated in some children suffering from severe infections (Ward & Schlegel 1969, Smith et a1 1972, Soriano et a1 1973), in chronic hypommplementaemic glomerulonephritis (Gewurz et a1 1967), and in hepatic cirrhosis (DeMeo & Andersen
1972). An inhibitor of chemotaxis is also present in low concentration in normal sera, and it is obviously one component of the homeostatic control system of chemotaxis (Berenberg & Ward 1973). In Hodgkin’s disease (Ward & Berenberg 1974) and in Bruton-type agammaglobulinaemia (Ward 1972) abnormal chemotaxis-inhibiting activity has been observed in the serum; this activity was probably caused by the physiological inhibitor in abnormally high concentration. There are few reports on serum inhibitors of phagocytosis. Penny & Galton (1966) demonstrated defective neutrophil phagocytosis in myelomatosis and macro-
28
T. RUUTU, P. RUUTU, P.VUOPI0, K. FRANSSILA & E. LINDER
globulinaemia, caused by inhibitory activity in plasma. In this paper we describe an inhibitor of chemotaxis and phagocytosis in a woman with reticulum cell sarcoma. CASE REPORT A 64-year-old housewife was referred to the University Central Hospital, Helsinki, in May 1970 because of lymphadenopathy and thrombocytopenia of several months’ duration. Enlarged lymph nodes, up t o 2 cm in diameter, were found in the neck, supraclavicular fossae, axillae and groins. The liver and the spleen were palpable. Slight normochromic anaemia and a normal leucocyte count with slight neutropenia were noted. The number of platelets was normal. In the bone marrow 10-20 % of the cells were atypical large blast cells, and the same cell type was also found in spleen aspirate. A lymph node biopsy specimen showed nonspecific follicular hyperplasia. Lymphography revealed enlargement of paraaortic lymph nodes. The patient was closely followed for the next 3 years. In autumn 1970 bleedings occurred and thrombocytopenia was observed. The life span of SICr-labelled platelets was one-third of the normal and splenectomy was performed. The spleen weighed 700 g. Some extramedullary haematopoiesis was seen microscopically, but nothing t o indicate malignancy. The enlarged lymph nudes at the hilus of the spleen were hyperplastic. The constant histological finding on repeated biopsies of the persistently enlarged lymph nodes was nonspecific hyperplasia. After the splenectomy the number of platelets was constantly normal. There was usually slight leucocytosis with a normal differential count. In bone marrow aspirates up t o 15 % atypical blast cells were seen at times. Bone marrow chromosome analysis was normal. The leucocyte alkaline phosphatase score was constantly low, 2 to 20. There was no lysozyme in the urine. The ESR (Westergren 1 h) varied between 6 and 28 mm. The serum gammaglobulins were increased, 17-25 g/l, and IgG, JgA and IgM were all polyclonally elevated. N o Bence Jones protein was found. The latex test and antinuclear antibodies were negative in the beginning, later positive. No LE-cells were seen. Tests for sarcoidosis, tuberculosis, mononucleosis, brucel-
losis, tularaemia, leptospirosis, yersinia infection, toxoplasmosis and listeriosis as well as screening for viral antibodies gave negative or normal results. The concentration of C‘3 was 0.76 g/], that of C’4 0.51 g/l. The lymphocyte response to phytohaemagglutinin was diminished. In lymphography the paraaortic lymph nodes were constantly enlarged. N o mediastinal lymph node enlargement was seen. Epigastric pains and loss of weight appeared in summer 1973. Gastroscopy revealed a cardiac tumour mass. Histologically the tumour was difficult to classify, but the mmt probable diagnosis was reticulum cell sarcoma. At laparotomy the tumour was found to extend as far as the hepatic flexure and far into the retroperitoneal space. No active surgery was performed. The patient died in August 1973, two days after the operation. At autopsy a flat tumour was found to encircle the distal part of the eosophagus and the cardia. Similar tumour growth was also found in the middle of the small intestine, the wall of which was perforated resulting in bacterial peritonitis. Histologically the tumour was a reticulum cell sarcoma. Microscopic examination of the enlarged paraaortic lymph nodes revealed nonspecific hyperplasia. No treatment, except occasional symptomatic medication, was given during the course of the disease because of the lack of diagnostic histological findings in the lymph node specimens. METHODS Phago cy tosis
This method has been described in detail elsewhere (Ruutu 1972). From fresh hepannized blood most of the erythrocytes were removed by Boyum’s method (1968). Cells separated from leucocyte-rich plasma were washed once with Hanks’ solution containing 1 % of bovine serum albumin and suspended in plasma, serum, or Hanks’ solution containing plasma or serum, as indicated. For the assay, Staphylococcus epidermidis, killed with formalin and washed with saline, was added to the leucocyte suspension in the relation 5 bacteria t o one neutrophil. The tubes were incubated at 37O C and mixed at intervals. After 6 min smears for microscopy were made and stained with Wright’s stain. Phagocytic index,
INHIBITOR OF CHEMOTAXIS AND PHAGOCYTOSIS the average number of bacteria phagocytosed by one neutrophil, was determined from 200 cells. Bactericidal activity of leucocytes
To measure the ability of leucocytes to kill Staphylococcus aureus, a slight modification (Ruutu 1972) of the method of Holmes et a1 (1966) was used
29
activity and assayed for IgG content and albumin content by radial immunodiffusion. For technical reasons fractions of patient and normal sera were tested on successive days with simultaneo’us controls.
RESULTS
Phagocytosis (Table I) Chemotaxis
A modification of the Boyden technique (1962) was used. The chamber was divided into an upper and a lower compartment by a membrane with an average pore diameter of 3.0 ,um (Millipore MF membranes). The chemotactic stimulus was prepared by incubating serum with zymosan (1 mg zymosan par 1 ml serum) for 1 h at 37O C, and diluted with Hank’s solution as indicated. Fresh frozen pooled serum from healthy donors was used as normal serum. The cells were suspended in Hanks’ solution containing 1 % of b e vine serum albumin at a concentration of 2 x lo6 granulocytes per ml. The chambers, containing 1 ml of chemotactic stimulus in the lower compartment and 1 ml of cell suspension in the upper compartment, were incubated at 37OC for 3 h. The filters were then removed, fixed in ethanol, stained with haematoxylin, dried, and cleared in xylene. The number of cells that had migrated through the filter t o the lower surface was counted at a magnification of 400 x in 10 random fileds. Chemotactic activity was expressed as the average number of cells per highpower field. All tests were done in triplicate. Serum fractionation
Partial purification of the factor@) responsible for the inhibition of chemotaxis and phagocytosis in the serum was achieved by column chromatography on Sephadex G-150 (Pharmacia) in 0.1 M Tris-HC1 buffer at pH 8.3. Pooled fractions to be tested for inhibiting activity were passed through a bacterial filter with pore size 0.22 ,U (Falcon plastics) and dialyzed against Hanks’ salt solution. The fractions were then concentrated by negative pressure dialysis t o a volume of 5 x the serum volume subjected t o fractionation. The concentrated fractions were tested for inhibitor
Phagocytosis by the patient neutrophils was deficient when the incubation medium contained patient plasma and normal in the presence of normal plasma. The same was true for cells from healthy controls: when plasma from the patient was used, phagocytosis was impaired. It is obvious that the inhibition of phagocytosis was plasmaassociated and that the cells were normal in their function. Serum behaved like plasma. When both patient and normal plasma were used in the phagocytosis assay, phagocytosis was impaired. This indicates that the defect of phagocytosis was caused by an inhibitor. If plasma lacking some factor necessary for phagocytosis is added to a system containing normal plasma, this may be expected to have no influence on phagocytosis or to increase it, since an increase of the plasma concentration enhances phagocytosis. The inhibition of phagocytosis was roughly the same in relation to the control whether 80 or 10 % of plasma was used in the incubation medium. Heating of the serum for 30 min at 5 6 O C did not affect the inhibition of phagocytosis. In our standard phagocytosis assay, mixing is not continuous in order to avoid clumping of cells. To exclude the possibility that this type of mixing would allow chemotaxis to influence the results of the phagocytosis tests, experiments were also made
T. RUUTU, P. RUUTU, P.VUOPl0, K. FRANSSILA & E. LINDER
30
Cells Plasma (or serum) concentration
I
I
Comment
PC PC NC NC
PP NP PP NP
80% 80% 80% 80%
PC PC PC PC NC NC NC NC
NP NP NP PP NP NP NP PP
20% 10% 1 0 % + PP 1 0 % 10% 20% 10% 10% + PP 1 0 % 10%
NC NC
PS 1 0 % NS 1 0 %
Similar defect in serum
NC NC NC NC
NP PP NP PP
80% 80% 10% 10%
Roughly unchanged inhibition with different plasma concentrations
NC NC NC NC
NS NS NS NS
80% 10 % + NS 70 % 30 min 5 6 O C l O % + P S 70% 10 % + PS 70 % 30 min 56O C
The inhibitor is heat-stable
The defect is plasma-associated
The defect is caused by an inhibitor
PI = phagocytic index, phagocytosed bacteria per one neutrophil. NC = normal cells, PC = patient cells, NP = normal plasma, PP = patient plasma, NS = normal serum, PS = patient serum.
with continuous mixing on an inclined wheel (not shown in the table). The inhibition of phagocytosis by patient plasma was at least as strong as in the standard assay. The inhibition of phagocytosis was demonstrated for the first time one and half years before the patient died, and several times thereafter. Bactericidal activity
After ingestion in the presence of normal serum, the ability of the patient’s leucocytes to kill Staphylococcus aureus was normal. Chemotaxis
Methods for the study of chemotaxis were
not available while the patient was alive. Therefore, deep-frozen (- 40° C) patient serum was used in the following investigations, which include no tests with patient cells. Table I1 shows a representative chemotaxis experiment. Patient serum in the cell compartment impaired chemotaxis. When bolth normal and patient serum were added, chemotaxis was also reduced. When patient serum was used as the source of chemotactic factors in the lower compartment, chemotaxis was highly deficient. Chemotaxis was also impaired when both normal and patient serum were used in the lower chamber. These experiments show that defective chemotaxis in the presence of patient se-
31
INHIBITOR OF CHEMOTAXIS AND PHAGOCYTOSIS TABLE I1 Chemotaxis (representative experiment) Cells Per field
Lower compartment
Cell compartment
NC, 10 % NS NC, 10 % NS NC, 10 % PS NC, 5 % NS + 5 % PS NC, 5 % NS NC, 10 % NS NC, 10 % NS NC, 10 % NS NC, 10 % NS NC, 10 % PS heated 30 min 56O C
30 % NS, heated 30 min 56O C 30 % NS, zymosan 30 % NS, zymosan 30 % NS, zymosan 30 % NS, zymosan 3 0 % PS, zymosan 20 % NS, zymosan 20 % NS, zymosan + 30 % NS 20 % NS, zymosan + 30 % PS 30 70NS, zymosan
2 38 3 27 44 1 36 39 21 14
% of positive control
5 100
\
7: 117 J
Comment
Negative control Positive control Inhibition of chemotaxis when patient serum in cell compartment Inhibition of chemotaxis when patient serum in lower compartment Inhibition of chemotaxis after heating of serum
NC = normal cells, NS = normal serum, PS = patient serum
rum was caused in part at least by an inhibitor. Patient serum was a poor source of chemotactic activity, but for this the inhibitor may be responsible; nothing definite can thus be said about the ability of the serum to produce chemoltactic factors. Inhibition of chemotaxis was also observed after heating of the patient’s serum at 56O C for 30 min. Eflect of serum fractions on chemotaxis and phagocytosis Chromatography on Sephadex G-150, V, 120 ml, yielded inhibitory activity for both chemotaxis and phagocytosis in the second of three major protein peaks, corresponding to 134-167 ml (Figure 1). IgG activity was also demonstrated in this part of the eluate. Separation from IgG activity was attempted by dividing the eluate into smaller fractions, but also in this experiment inhibitor activity coincided with the peak of IgG activity. The higher protein concentration in the inhibitory fraction as compared with the corresponding control fraction does not ex-
plain the inhibition, at least not the inhibition of phagocytosis, since an increased concentration of serum or plasma in the assay tends to enhance phagocytosis (Table I). DISCUSSION
The present experiments show that the patient had a serum inhibitor of chemotaxis. Such an inhibitor may affect phagocytic cells (Ward & Schlegel 1969, Smith et a1 1972, Soriano et a1 1973) or chemotactic factors (Gewurz et a1 1967, DeMeo & Andersen 1972, Ward 1972, Berenberg & Ward 1973, Ward & Berenberg 1974). No definite conclusions can be drawn about the point of action of the present inhibitor. In experiments reported by others, a celldirected inhibitor was active only in the cell compartment but not in the lower compartment (Smith et a1 1972, Soriano et a1 1973), whereas a chemotactic factordirected inhibitor was active in low concentrations in the lower compartment only, in high concentrations in both compartments (Berenberg & Ward 1973). In these
32
T. RUUTU, P. RUUTU, P.VUOPI0, K. FRANSSILA & E. LINDER
experiments the effect was of roughly the same magnitude whether patient serum was added into the cell compartment or into the lower compartment. It is not surprising that an inhibitor exerts its effect independent of which compartment it is introduced into, since the filter does not hinder diffusion. It may be assumed that a chemotactic factor inhibitor in particular could be
effective even when introduced into the cell compartment, considering that the chemotactic factors must diffuse through the filter to exert their action. In the inhibition of phagocytosis, a humoral object of action seems probable, since the reduction of the ratio of plasma to cells to 1:8 did not diminish the inhibition. The cell-directed inhibitors of chemotaxis have always been
1
- 100 g
E
'ij
%.
.* 50
I
serum
I
5
Patient serum
I
04u=.-I__--.Lp
om
N
111
150
200
V
-
250
,
300
350
VI
Fraction
403
Effluentvolume ml
Figure 1. Effect of fractions of patient and normal sera on phagocytosis and chemotaxis of normal neutrophils Phagocytosis assay Chemotaxis assay Cell compartment Control Fractions Control Fractions 5 x lo6 neutrophils 5 x 106 neutrophils 2 x 106 neutrophils 2 x lo6 neutrophils 0.8 ml Hanks 0.8 ml fraction 0.9 ml Hanks + 0.5 ml Hanks + 0.1 ml normal serum 0.1 ml normal serum 0.5 % BSA 0.5 % BSA 0.1 ml bacterial 0.1 ml bacterial 0.1 ml normal serum 0.5 ml fraction suspension suspension Lower compartment 0.3 ml normal serum + zymosan 0.7 ml Hanks PI = phagocytic index
BSA = bovine serum albumin
INHIBITOR OF CHEMOTAXIS AND PHAGOCYTOSIS
associated with severe susceptibility to infections (Ward & Schlegel 1969, Smith et a1 1972, Soriano et a1 1973), but in patients with chemotactic factor inhibitor (Gewurz et a1 1967, DeMeo & Andersen 1972, Ward & Berenberg 1974) the tendency has not been so striking. The present patient was not exceptionally susceptible to infections. Therefore, inhibition of chemotactic factors seems more probable in this case. Although various agents have been shown to inhibit phagocytosis in vivo and in vitro in experimental conditions, there are very few reports on serum inhibitors of phagocytosis in patients. Penny & Galton (1966) demonstrated phagocytosis-inhibiting activity in the plasma of patients with myelomatosis and macroglobulinaemia. This activity was correlated with the concentration of M-protein. It was suggested that M-protein is adsorbed on the surface of neutrophils, and interferes with membrane potential or with the action of serum factors necessary for phagocytic ability. Sbarra et a1 (1964) described impaired phagocytic capability of leucocytes in a few patients with lymphosarcoma and chronic lymphocytic leukaemia. In some of these patients the defect could be corrected, as in the present case, by substituting normal serum for autologous serum. No effort was made to study, whether the serum-associated defect was due to the deficiency of some factor or an inhibitor. Combined defects of chemottaxis and phagocytosis have been described before (Miller & Nilsson 1970, McCall et a1 1971, Miller 1971, Steerman et a1 1971). This is not surprising considering the interaction of mechanisms (Stossel 1974). In the present case the inhibitory activities for chemotaxis and phagocytosis were found in the same serum fraction, and the same Scand J Haernatol(1975) 15
33
factor was probably responsible for both. The effects of previously described inhibitors of chemotactic factors (Gewurz et a1 1967, DeMeo & Andersen 1972, Ward & Berenberg 1974) on phagocytosis were not tested. As the inhibitory activities could not be separated from IgG by chromatography, the inhibitor may be an immunoglobulin. Further efforts to characterize the inhibitor, e.g., by ion-exchange chromatography, were prevented by a shortage of serum post mortem. The inhibitor may be an autoantibody against some humoral factor associated with chemotaxis and phagocytosis. Autoantibodies occur in malignant diseases of the lymphatic system (Seligmann et a1 1965, Sacks 1974). The mechanism of inhibition seems to be different from that in myelomatosis and macroglobulinaemia (Penny & Galton 1966). In those diseases, the inhibition is correlated, in part at least, with the concentration of M-protein, and is probably caused by this protein, as stated above. In this patient no M-protein was found. Why the disorder, which was quite severe in vitro, did not clinically affect the resistance to infection remains obscure. This investigation was supported by the Sigrid Jus6lius Foundation, Helsinki. REFERENCES Berenberg J L & Ward P A (1973) Chemotactic factor inactivator in normal human serum. J Clin Invest 52, 1200-06. Boyden S (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med 115, 453-66. Boyum A (1968) Isolation of leucocytes from human blood. Scand J Clin Lab Invest 21, Suppl. 97, 9-29. DeMeo A N & Andersen B R (1972) Defective 3
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T. RUUTU, P. RUUTU, P.VUOPI0, K. FRANSSILA & E. LINDER
chemotaxis associated with a serum inhibitor in cirrhotic patients. N Engl J Med 286, 735-40. Gewurz H, Page A R, Pickering R J & Good R A (1967) Complement activity and inflammatory neutrophil exudation in man. Studies in patients with glomerulonephritis, essential hypocomplementemia and agammaglobulinemia. Int Arch Allergy Appl lmmunol 32, 64-90. Holmes B, Quie P G, Windhorst D B & Good R A (1966) Fatal granulomatous disease of childhood. An inborn abnormality of phagocytic function. Lancet 1, 1225-28. McCall C E, Caves J, Cooper R & DeChatelet L (1971) Functional characteristics of human toxic neutrophils. J Infect Dis 124, 68-75. Miller M E (1971) Chemotactic function in the human neonate: humoral and cellular aspects. Pediat Res 5, 487-92. Miller M E & Nilsson U R (1970) A familial deficiency of the phagocytosis-enhancing activity of serum related t o a dysfunction of the fifth component of complement (C5). N Engl J Med 282, 354-58. Penny R & Galton D A G (1966) Studies on neutrophil function. 11. Pathological aspects. Br J Haematol 12, 633-45. Ruutu T (1972) Effect of phenothiazines and related compounds on phagocytosis and bacterial killing by human neutrophilic leukocytes. Ann Med Exp Biol Fenn 50, 24-36. Sacks P V (1974) Autoimmune hematologic complications in malignant lymphoproliferative disorders. Arch Intern Med 134, 781-83. Sbarra A J, Shirley W, Selvaraj R J, Ouchi E & Rosenbaum E (1964) The role of the phagocyte
in host-parasite interactions. I. The phagocytic capabilities of leukocytes from lymphoproliferative disorders. Cancer Res 24, 1958-68. Seligmann M, Cannat A & Hamard M (1965) Studies on antinuclear antibodies. Ann N Y Acad Sci 124, 816-32. Smith C W, Hollers J C, Dupree E, Goldman A S & Lord R A (1972) A serum inhibitor of leukotaxis in a child with recurrent infections. J Lab Clin Med 79, 878-85. Soriano R B, South M A, Goldman A S & Smith C W (1973) Defect of neutrophil motility in a child with recurrent bacterial infections and disseminated cytomegalovirus infection. J Pediatr 83, 951-58. Steerman R L, Snyderman R, Leikin S L & Colten H R (1971) Intrinsic defect of the polymorphonuclear leucocyte resulting in impaired chemotaxis and phagocytosis. Clin Exp lmmunol 9, 939-46. Stossel T P (1974) Phagocytosis. N Engl J Med 290, 833-39. Ward P A (1972) Natural and synthetic inhibitors of leukotaxis. In I H Lepow & P A Ward (eds) Inflammation Mechanisms and Control, pp 30108. Academic Press, New York. Ward P A & Berenberg J L (1974) Defective regulation of inflammatory mediators in Hodgkin's disease. Supernormal levels of chemotactic-factor inactivator. N Engl J Med 290, 7680. Ward P A & Schlegel R J (1969) Impaired leucotactic responsiveness in a child with recurrent infections. Lancet 2, 344-47.
