Immunology Letters, 24 (1990) 113-116
Elsevier IMLET01370
Neutrophil-mediated cytotoxicity induced by secretory IgA J. R. Geffner, E Minnucci and M. A. Isturiz Seci6n Inmunologia, Instituto de Investigaciones Hematoldgicas, Academia Nacional de Medicina, Buenos Aires, Argentina
(Received 17 January 1990; accepted25 January 1990)
1. Summary
Normal human neutrophils triggered by secretory IgA (slgA) displayed low levels of cytotoxicity towards non-sensitized red blood cells. Catalase completely impaired this non-specific cytotoxicity (NSC), while superoxide dismutase (SOD) significantly enhanced it, suggesting a key role for hydrogen peroxide (H202) in the lysis of target cells. Three heme-enzyme inhibitors, sodium azide, sodium cyanide and 3-amino-l,2,4-triazole, did not decrease NSC, but significantly enhanced it, suggesting that the mechanism involved is not dependent upon myeloperoxidase (MPO). Heat-aggregated IgG (HA-IgG) synergize with slgA in promoting NSC. It was also found that gamma interferon significantly enhanced neutrophil-mediated NSC induced by slgA, its effect being more dramatic on NSC triggered by low concentrations of slgA. The significance of these results is discussed. 2. Introduction
The presence of receptors for the Fc portion of IgA (Fco~R)on lymphocytes [1- 3], monocytes [4, 5] and polymorphonuclear leukocytes (PMN) [4, 5] has now been established. It would appear that FcaR can react with both the IgA1 and IgA2 subclasses, with secretory IgA (slgA) and with both monomer and dimer forms of IgA [6]. While lymphocyte receptors for IgA may be associated with immuno-
regulatory mechanisms [7], it seems likely that their presence on phagocytes may be related to the induction of effector functions [6]. In fact, PMN are capable of phagocytosing IgA-coated target cells [4, 6]. Furthermore, IgA-immune complexes are able to induce degranulation of primary and secondary granules [8] and superoxide production by PMN [9, 10]. We have previously showed that the stimulation of neutrophils with IgG-immune complexes enabled them to damage non-sensitized target ceils through a mechanism known as non-specific cytotoxicity (NSC) [11, 12]. In the present study, we report the ability of slgA-triggered neutrophils to mediate NSC by secretion of H20 2, which acted independently of the myeloperoxidase (MPO) system. 3. Materials and Methods 3.1. E f f e c t o r cells
Peripheral blood PMN were isolated from heparin-treated human blood samples by FicollHypaque centrifugation [13] and sedimentation in dextran. Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells were resuspended in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 5°70 heat-inactivated fetal calf serum (Difco, Detroit, MI) and 50 #g/ml gentamicin (TCM), to a final concentration of 2x106 PMN/ml. Aliquots of 0.1 ml of this suspension, containing 95-98°70 neutrophils, were placed in 96-well flat-bottomed microtiter plates.
Key words." Neutrophilcytotoxicity;SecretoryIgA Correspondence to: Jorge Ratll Geffner,Secci6nInmunologfa,
Institutode InvestigacionesHematol6gicas,AcademiaNacional de Medicina,Pachecode Melo3081,(1425)BuenosAires,Argentina. FAX:(541)805-3415.
3.2. Reagents
slgA, isolated from the pooled human colostrum, was obtained from Sigma Chemical Co. (St. Louis,
0165-2478 / 90 / $ 3.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
113
MO). It was further purified to eliminate traces of IgG by passage through a protein G Sepharose column (Pharmacia, Uppsala, Sweden). The sIgA preparation was shown by ELISA methods to contain less than 0.1% IgG on a weight basis. Heataggregated IgG (HA-IgG) were prepared by heating human IgG (Sigma) for 12 min at 63 °C. The IgG aggregates were then centrifuged at 300×g for 15 min and the precipitate was discarded. Catalase (bovine liver, type V, 3090 U/mg protein), superoxide dismutase (SOD) (3300 U/mg protein) and 3-amino1,2,4-triazole were obtained from Sigma; sodium azide and sodium cyanide from Fisher Scientific Co. (Fair Lawn, USA); recombinant human gamma interferon fom Serono (Israel). 3.3. Non-specific cytotoxicity induced by slgA Unless otherwise stated, sIgA was added to 2×105 neutrophils in a final volume of 0.1 ml, 5 min after the addition of 2×105 non-sensitized 51Cr-labeled chicken red blood cells (CRBC). The plates were incubated for 14 h at 37°C in 5% CO2/95% humidified air. After centrifiguation of the culture plates, the radioactivity of the supernatant and the pellet was measured and the percentage of lysis calculated. 3.4. Statistics The statistical significance of results was calculated using Student's t-test.
4. Results
4.1. Neutrophil-mediated non-specific cytotoxicity induced by sIgA Experiments were designed to study the ability of sIgA to induce the lysis of non-sensitized red blood cells. As shown in Table 1, slgA-triggered neutrophils were able to induce the lysis of target cells in a dose-related fashion. However, there were significant differences in NSC percentages obtained with different neutrophil donors. In fact, when we studied the NSC carried out by 2× 105 neutrophils triggered by 50/~g/ml slgA (E:T ratio 1:1), we found that 4 out of 21 donors tested showed no significant level of cytotoxicity (%o NSC = 4.7_+2.3, mean _+ s.e., N=4). 4.2. Role of oxidative metabolism in NSC induced
by slgA The role of oxidative metabolism in NSC was evaluated employing the oxygen metabolite scavengers catalase and superoxide dismutase (SOD). As shown in Table 2, catalase completely impaired NSC while SOD significantly enhanced it ( P < 0.01), suggesting that H202 plays a key role in NSC. H202 could directly mediate the lysis of target cells or could serve as a component of the H202halide-MPO system [14, 15]. In order to study the involvement of this system in NSC, the heme-enzyme inhibitors azide, cyanide and 3-amino-l,2,4-triazole [13, 14] were used. All these compounds did not decrease NSC; on the contrary, they significantly enTABLE 2 Role of oxidative metabolism in NSC induced by sIgA.
TABLE 1 Treatment
% NSC
None Catalase 1000 U / m l Heated catalase 1000 U / m l SOD 1000 U / m l Azide 1.0 mM Amino-triazole t .0 mM Cyanide 1.0 mM
24.7 _+4.0 3.3 _+ 1.8 21.4+_ 3.6 32.2+_4.2 38.8_+3.3 46.5 +- 5.1 30.2 _+3.8
Neutrophil-mediated NSC induced by slgA. slgA (/~g/ml)
% NSC
None 10 20 50 200
2.3 +- 1.0 8.4_+2.4 19.6+-4.4 27.3 +_4.1 25.6 +_3.5
Data are expressed as the mean _+ s.e. of triplicates of nine different donors.
114
Catalase was heat-inactivated at 100°C for 15 min. NSC was triggered by 50/~g/ml of slgA. Data are expressed as the mean _+ s.e. of triplicates of seven different donors.
hanced it (P
Elsevier IMLET01370
Neutrophil-mediated cytotoxicity induced by secretory IgA J. R. Geffner, E Minnucci and M. A. Isturiz Seci6n Inmunologia, Instituto de Investigaciones Hematoldgicas, Academia Nacional de Medicina, Buenos Aires, Argentina
(Received 17 January 1990; accepted25 January 1990)
1. Summary
Normal human neutrophils triggered by secretory IgA (slgA) displayed low levels of cytotoxicity towards non-sensitized red blood cells. Catalase completely impaired this non-specific cytotoxicity (NSC), while superoxide dismutase (SOD) significantly enhanced it, suggesting a key role for hydrogen peroxide (H202) in the lysis of target cells. Three heme-enzyme inhibitors, sodium azide, sodium cyanide and 3-amino-l,2,4-triazole, did not decrease NSC, but significantly enhanced it, suggesting that the mechanism involved is not dependent upon myeloperoxidase (MPO). Heat-aggregated IgG (HA-IgG) synergize with slgA in promoting NSC. It was also found that gamma interferon significantly enhanced neutrophil-mediated NSC induced by slgA, its effect being more dramatic on NSC triggered by low concentrations of slgA. The significance of these results is discussed. 2. Introduction
The presence of receptors for the Fc portion of IgA (Fco~R)on lymphocytes [1- 3], monocytes [4, 5] and polymorphonuclear leukocytes (PMN) [4, 5] has now been established. It would appear that FcaR can react with both the IgA1 and IgA2 subclasses, with secretory IgA (slgA) and with both monomer and dimer forms of IgA [6]. While lymphocyte receptors for IgA may be associated with immuno-
regulatory mechanisms [7], it seems likely that their presence on phagocytes may be related to the induction of effector functions [6]. In fact, PMN are capable of phagocytosing IgA-coated target cells [4, 6]. Furthermore, IgA-immune complexes are able to induce degranulation of primary and secondary granules [8] and superoxide production by PMN [9, 10]. We have previously showed that the stimulation of neutrophils with IgG-immune complexes enabled them to damage non-sensitized target ceils through a mechanism known as non-specific cytotoxicity (NSC) [11, 12]. In the present study, we report the ability of slgA-triggered neutrophils to mediate NSC by secretion of H20 2, which acted independently of the myeloperoxidase (MPO) system. 3. Materials and Methods 3.1. E f f e c t o r cells
Peripheral blood PMN were isolated from heparin-treated human blood samples by FicollHypaque centrifugation [13] and sedimentation in dextran. Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells were resuspended in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 5°70 heat-inactivated fetal calf serum (Difco, Detroit, MI) and 50 #g/ml gentamicin (TCM), to a final concentration of 2x106 PMN/ml. Aliquots of 0.1 ml of this suspension, containing 95-98°70 neutrophils, were placed in 96-well flat-bottomed microtiter plates.
Key words." Neutrophilcytotoxicity;SecretoryIgA Correspondence to: Jorge Ratll Geffner,Secci6nInmunologfa,
Institutode InvestigacionesHematol6gicas,AcademiaNacional de Medicina,Pachecode Melo3081,(1425)BuenosAires,Argentina. FAX:(541)805-3415.
3.2. Reagents
slgA, isolated from the pooled human colostrum, was obtained from Sigma Chemical Co. (St. Louis,
0165-2478 / 90 / $ 3.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
113
MO). It was further purified to eliminate traces of IgG by passage through a protein G Sepharose column (Pharmacia, Uppsala, Sweden). The sIgA preparation was shown by ELISA methods to contain less than 0.1% IgG on a weight basis. Heataggregated IgG (HA-IgG) were prepared by heating human IgG (Sigma) for 12 min at 63 °C. The IgG aggregates were then centrifuged at 300×g for 15 min and the precipitate was discarded. Catalase (bovine liver, type V, 3090 U/mg protein), superoxide dismutase (SOD) (3300 U/mg protein) and 3-amino1,2,4-triazole were obtained from Sigma; sodium azide and sodium cyanide from Fisher Scientific Co. (Fair Lawn, USA); recombinant human gamma interferon fom Serono (Israel). 3.3. Non-specific cytotoxicity induced by slgA Unless otherwise stated, sIgA was added to 2×105 neutrophils in a final volume of 0.1 ml, 5 min after the addition of 2×105 non-sensitized 51Cr-labeled chicken red blood cells (CRBC). The plates were incubated for 14 h at 37°C in 5% CO2/95% humidified air. After centrifiguation of the culture plates, the radioactivity of the supernatant and the pellet was measured and the percentage of lysis calculated. 3.4. Statistics The statistical significance of results was calculated using Student's t-test.
4. Results
4.1. Neutrophil-mediated non-specific cytotoxicity induced by sIgA Experiments were designed to study the ability of sIgA to induce the lysis of non-sensitized red blood cells. As shown in Table 1, slgA-triggered neutrophils were able to induce the lysis of target cells in a dose-related fashion. However, there were significant differences in NSC percentages obtained with different neutrophil donors. In fact, when we studied the NSC carried out by 2× 105 neutrophils triggered by 50/~g/ml slgA (E:T ratio 1:1), we found that 4 out of 21 donors tested showed no significant level of cytotoxicity (%o NSC = 4.7_+2.3, mean _+ s.e., N=4). 4.2. Role of oxidative metabolism in NSC induced
by slgA The role of oxidative metabolism in NSC was evaluated employing the oxygen metabolite scavengers catalase and superoxide dismutase (SOD). As shown in Table 2, catalase completely impaired NSC while SOD significantly enhanced it ( P < 0.01), suggesting that H202 plays a key role in NSC. H202 could directly mediate the lysis of target cells or could serve as a component of the H202halide-MPO system [14, 15]. In order to study the involvement of this system in NSC, the heme-enzyme inhibitors azide, cyanide and 3-amino-l,2,4-triazole [13, 14] were used. All these compounds did not decrease NSC; on the contrary, they significantly enTABLE 2 Role of oxidative metabolism in NSC induced by sIgA.
TABLE 1 Treatment
% NSC
None Catalase 1000 U / m l Heated catalase 1000 U / m l SOD 1000 U / m l Azide 1.0 mM Amino-triazole t .0 mM Cyanide 1.0 mM
24.7 _+4.0 3.3 _+ 1.8 21.4+_ 3.6 32.2+_4.2 38.8_+3.3 46.5 +- 5.1 30.2 _+3.8
Neutrophil-mediated NSC induced by slgA. slgA (/~g/ml)
% NSC
None 10 20 50 200
2.3 +- 1.0 8.4_+2.4 19.6+-4.4 27.3 +_4.1 25.6 +_3.5
Data are expressed as the mean _+ s.e. of triplicates of nine different donors.
114
Catalase was heat-inactivated at 100°C for 15 min. NSC was triggered by 50/~g/ml of slgA. Data are expressed as the mean _+ s.e. of triplicates of seven different donors.
hanced it (P
