Trun.$isioii Medicine. 1992. 2, 283-287
Location of the granulocyte-specific antigen LAN on the Fc-receptor I11 P. Metcalfe and A. H. Waters Deparrmenr of
Haemalology. Si Barrholornew’s Hospiral and Medical College,
London E C l A 7BE. U.K. Receiced 9 January 1992; arcepred for publicarion 18 June 1992
~
This report investigates the location of the binding site of a recently described granulocytespecific alloantibody, designated LAN, which causes alloimmune neonatal neutropenia. The LAN antigen
SUMMARY.
Granulocyte-specific alloantibodies are clinically important in neonatal alloimmune neutropenia, nonhaemolytic febrile transfusion reactions (Minchinton & Waters, 1984) and transfusion-related acute lung injury (Yomtovian el al., 1984). Of the granulocytespecific alloantigens described so far (McCullough el al., 1987). only the NAI/NA2 and NBI antigens have been found to be located on particular glycoproteins (Werner e f al., 1985; Huizinga e f al., 1990a; Stroncek e f al., 1990). Recently, a new granulocyte-specific alloantibody, designated LAN, was described in an Australian Aboriginal mother of a baby with neonatal alloimmune neutropenia (Rodwell et al., 1991). This report describes the location of the binding site of the LAN antibody on the Fc-receptor 111 (FcRIII).
MATERIALS A N D METHODS LAN antibody The LAN antibody is a granulocyte-specific alloantibody which recognizes a previously undefined antigen present at high frequency ( > 99%) in predominantly Caucasian populations (Rodwell et al., 1991). The antibody is of the IgG class and reacts strongly in immunofluorescence and agglutination techniques. Monoclonal antibodies
The following mAb were used: anti-FcRIII (CLBFcRgran/l),anti-NA I (CLBgran/l I), both supCorrcspondence: Dr P. Metcalfe. Department of Haematology. SI Barrholomew’s Hospital and Medical College. West Smithfield. London EClA 7BE. U . K .
is shown to be situated on the Fc-receptor I11 (FcRIII). Key words: alloimmune neonatal neutropenia, FcRIII, granulocyte alloantibody, LAN antibody.
plied by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, and anti-Gp IIb/IIIa (NIB 85/661), supplied by The National Institute for Biological Standards and Control, Potters Bar. U.K.
Granulocyte separafion andfixation
Granulocytes were isolated from freshly collected, EDTA-anticoagulated, peripheral blood (1 volume 5 % Na2 EDTA:9 volumes blood). Two millilitres of dextran solution [5% dextran, Sigma, average mot. wt 162.000, in phosphate-buffered saline (PBS)] were mixed with each 10 ml of blood. After incubation at 37°C for 30 min the leucocyte-rich supernatant was removed and 2 ml were underlayered with 2 ml lymphocyte separation medium (LSM) (Ficoll-hypaque sp. gr. 1.077, Flow Laboratories) and 2 ml Monopoly resolving medium (MPRM) (Ficoll-hipaque sp. gr. 1.114, Flow Laboratories) in a 10-ml conical centrifuge tube. After centrifugation at 2,500 g for 5 min. the granulocytes were removed from the LSM/ MPRM interface and washed three times (400g, 5 min) in PBS/BSA (0.2%). The cells were either resuspended in PBS/BSA nr fixed in 1 % para-formaldehyde (5 min at room temperature) and washed twice more in PBS/ BSA and used as described below. Lymphocytes, used in preliminary studies to demonstrate the specificity of MAIGA reactions, were obtained from the leucocyte-rich supernatant/LSM interface at the same time as the granulocyte preparation and were processed in the same way as granulocytes.
284
P . Melcalfe and A . H . Waters
Granulocyte immunofluorescence tesl (GIFT)
Serum LAN and anti-FcRIII mAb were tested against granulocytes from a patient with paroxysmal nocturnal haemoglobinuria (PNH) and a normal donor, using the granulocyte immunofluorescence test (Verheugt et al., 1977). Pooled AB serum from 10 nontransfused male donors was used as a negative control and serum containing strong multispecific HLA antibodies was used as a positive control. Results were assessed by fluorescence microscopy using a scoring +) system (negative, weak, and the percentage of fluorescing cells was calculated by counting 300 cells.
+, + +, + + +, + + +
Monoclonal anribody-specijc immobilization granulocyte antigens ( M A I G A ) assay
00'
This assay (Fig. 1) was developed directly from the MAIPA assay for platelet antibodies described by Kiefel el al. (1987). Briefly, a pellet of 0.5-1.0 x lo6 granulocytes was resuspended in 30 pl human serum or plasma to be tested and incubated at 37°C for 30 rnin in a U-well microplate. After washing twice in PBS/BSA the granulocytes were resuspended in 30 pl mouse mAb, as detailed above, and incubated at 37°C for 30 min. The cells were then washed twice more in PBS/ BSA and lysed by the addition of 100 pI Tris buffered saline (TBS) containing 0.5% Nonidet P-40 and incubated at 4°C for 30 min. The granulocyte lysate was then transferred to a 2-ml conical tube and centrifuged at 11,600 g for 30 rnin at 4°C to remove
particulate matter. Sixty microlitres of the resulting supernatant were removed and diluted with 180pl TBS wash buffer (0.5%Nonidet P-40,0.05% Tween 20 and 0.5 mmol CaC12). One hundred microlitres of diluted granulocyte lysate were transferred, in duplicate to a flat-well microplate coated with goat anti-mouse IgG [prepared by overnight incubation of 100 p1 goat antimouse IgG (sera-tab, code SBA 1030-01)at 3 pg/ml in carbonate coating buffer, pH 9.6, followed by four washes and 30 rnin blocking in TBS wash buffer]. After incubation at 4°C for 90 min the microplate wells were washed four times with 200 pl TBS wash buffer followed by the addition of 100 pl alkaline phosphatase labelled anti-human IgG (Jackson, code 109-055008) diluted 1 :3000 in TBS wash buffer. After further incubation at 4°C for 90 rnin the wells were washed four times in TBS wash buffer and 100 p1 substrate solution [ 1 mg/ml p-nitrophenyl phosphate (Sigma) in diethanolamine buffer, pH 9.81 were added to each well. The resulting colour change was measured at 30 rnin using a dual wavelength photometer (Bio-Rad model 450). Results were expressed as the mean absorbance at 405 nm of duplicate tests minus the mean of eight blanks containing TBS wash buffer instead of granulocyte lysate. Pooled AB serum was used as a negative control. Anti-NA2 was used as a positive control in the MAIGA assays utilizing antiFcRIII mAb, but human NAI or NA2 antibodies could not be used as a positive control in the MAIGA assays utilizing anti-NAI mAb because of mutual inhibition. In some experiments lymphocytes were used in exactly the same way as granulocytes.
enzyme-labelled goat anll-human IgG
goat anti-mouse IgG
microplate
Fig. 1. Representation of the MAlGA assay.
Locarion of the granulocyte-specrfic antigen LAN
285
Table 1. GIFT results of anti-FcRIII
mAb and LAN antibody tested against granulocytes from a PNH patient and a normal donor, measured as a percentage
PNH patient
Antibody
Weakor negative
Anti-FcRIII mAb LAN antibody Negative control (AB serum) Positive control (anti-HLA)
73 74 100 0
RESULTS Immunojhorescence studies on PNH granulocytes Granulocytes were obtained from a 42-year-old woman who had paroxysmal nocturnal haemoglobinuria (PNH). Only 27% of the granulocytes showed strongly positive reactions when tested with antiFcRIII mAb, the remainder giving mostly weak positive or occasionally negative reactions, compared to 97% strongly positive reactions with normal donor granulocytes (Table I). The LAN antibody showed the same pattern of reactions when tested with granulocytes from the PNH patient, 26% of the cells were strongly positive and the remaining 74% of cells showed weak or negative reactions, while 98% of normal donor granulocytes were strongly positive. This weaker reaction pattern seen with PNH granulocytes was not seen in over 600 normal donors tested with the LAN antibody (Rodwell et al., 1991). These reactions suggest that the binding site of the LAN antibody is located on a membrane protein which is deficient in PNH cells. MAIGA studies The specificity of the MAIGA assay was shown by reactions of the LAN antibody with granulocytes and lymphocytes in tests using three different mAbs (Table 2). Positive reactions were obtained only when Table 2. Specificity of MAIGA assay
Immobilizing mAb Antibody
Cells
IIb/IIIa
FcRIlI
NAI
LAN LAN
Lymphocytes Granulocytes
0.020* 0.029
0.065 0.557
0.02 I 0.566
Values are mean absorbance of duplicate tests on each of donors.
IWO
Normal donor
+ + + or
++++ 27 26 0 100
Weakor negative 3 2 100 0
+ + + or ++++ 97 98 0 100
the LAN serum was tested against granulocytes in combination with either anti-FcRIII or anti-NA1 mAbs. All reactions against lymphocytes and reactions against granulocytes in combination with an irrelevant monoclonal antibody (anti-GP IIb/IIIa) were negative. The LAN alloantibody was tested against PFAfixed and unfixed granulocytes using both anti-FcRIII and anti-NAI mAb to immobilize the FcRIII receptor in the MAIGA. Positive results were obtained with both mAb (Fig. 2a and b), indicating that the LAN antigen is on the FcRIII receptor. Differentiation between positive and negative results was greater when PFA-fixed granulocytes were used. DISCUSSION
In contrast to platelet and red cell antigens, little is known about the location of the antigens recognized by granulocyte alloantibodies. The neutrophil-specific alloantigens NAI and NA2 have been localized on FcRIII (Werner et al., 1985; Huizinga et al., 1990a) and the NBI antigen has been demonstrated on a 5864 kDa glycoprotein present on the neutrophil membrane and in secondary granules (Stroncek et al., 1990). This report demonstrates that the LAN antigen is situated on FcRIII. This was suspected after immunofluorescence studies on PNH granulocytes showed that the LAN antibody gave the same reaction pattern as anti-FcRIII mAb. The reactions with PNH granulocytes were typical of those seen by van der Schoot et al. (1990) when they described the reactions of granulocytes from 20 patients with PNH tested in a cytofluorimetric assay with antibodies against glycosyl-phosphatidylinositol (GP1)-linked membrane proteins, including FcRIII. An antigen capture assay (MAIGA) was developed to study this further. FcRIII was immobilized in microplates by anti-FcRIII and -NAI mAbs and binding of the LAN antibody to FcRIII was demonstrated by ELISA. PFA-fixation of granulocytes resulted in better differentiation between positive and
P . Metcalfe and A . H . Waters
T, 0.5
1
that the LAN antibody was an iso-antibody against FcRIII formed in an “A-null’ mother (Huizinga et al., 1990b) was excluded by re-testing the mother and showing that her neutrophils were NA 1/NA2 positive (R. Minchinton, personal communication).
ACKNOWLEDGEMENTS We wish to thank Dr R. Rodwell for providing the LAN antibody, Dr R. Minchinton for typing the LAN neutrophils and Dr J. A. L. Amess who provided blood samples from the patient with PNH. Unfixed granulocytes
PFA.fixed granulocytes
REFERENCES
0.4
0.3
02
0.1
00
Unfixed granuiaytes
PFA-fixed granulocytes
Fig. 2. Reactions of the LAN antibody with PFA-fixed and unfixed donor granulocytes in the MAIGA assay, using, (a) anti-FcRIII mAb and granulocytes from four NA2 positive donors, (b) anti-NAl mAb and granulocytes from three NAI positive donors. Each column represents the mean of duplicate measurements on four donors and three donors, respectively, and the error bars indicate 1 standard deviation from the mean. (M)Negative control, (R)LAN antibody, (0)positive control.
negative results, as also shown for lymphocytes in the monoclonal antibody-specific immobilization of leucocyte antigens (MAILA) assay (Mueller-Eckhardt er al., 1989). Now that many anti-granulocyte mAb are available, the MAIGA technique may be useful in the investigation of other granulocyte allo- and autoantibodies. The LAN antigen is the second granulocyte-specific alloantigen to be located on FcRIII. The possibility
Huizinga, T.W.J., Kleijer, M., Tetteroo, P.A.T., Roos, D. & von dem Borne, A.E.G.Kr. (l990a) Biallelic neutrophil NA-antigen system is associated with a polymorphism on the phospho-inositol-linked Fcy receptor 111 (CD16). Blood, 75,213-217. Huizinga, T.W.J., Kuijpers, R.W.A.M., Kleijer, M., Schulpen, T.W.J., Cuypers, H.T.M., Roos, D. & von dem Borne, A.E.G.Kr. (1 990b) Maternal genomic neutrophil FcRIII deficiency leading to neonatal isoimmune neutropenia. Blood, 76, 1927-1932. Kiefel, V., Santoso, S., Weisheit, M. & Mueller-Eckhardt, C. (1987) Monoclonal antibody specific immobilisation of platelet Antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies. Blood, 70, 1722-1726. McCullough, J., Clay, M. & Kline, W. (1987) Granulocyte antigens and antibodies. Transfusion Medicine Reviews, 1, 150- 160. Minchinton, R.M. & Waters, A.H. (1984) The Occurrence and significance of neutrophil antibodies. British Journal of Haematology, 56, 521-528. Mueller-Eckhardt, G.,Kiefel, V., Schmidt, A., llusty, A., Santoso. S. & Mueller-Eckhardt, C. (1989) Discrimination of antibodies against antigens of different M H C loci in human sera by monoclonal antibody-specific immobilisation of leukocyte antigens. Human Immunology, 25, 125134. Rodwell, R.L., Tudhope, D.I., ORegan, P.O., Minchinton, R. &Waters, A.H. (1991) Alloimmune neonatal neutropenia in Australian aboriginals: an unrecognised disorder? Tramfusion Medicine, 1, 63-67. Stroncek, D.F., Skubitz, K.M. & McCullough, J.J. (1990) Biochemical characterization of the neutrophil-specific antigen NBI. Blood, 75, 744-755. van der Schoot, C.E., Huizinga, T.W.J., van’t Veer-Korthof, E.T., Wijmans, R., Pinkster, J. & von dem Borne, A.E.G. Kr. ( 1 990) Deficiency of glycosyl-phosphatidylinositollinked membrane glycoproteins of leucocytes in paroxysmal nocturnal haemoglobinuria, description of a new diagnostic cytofluorometric assay. Blood, 76, 1853-1 859.
Location of the granulocyte-specific antigen L A N Verheught, F.W.A., vondem Borne, A.E.G. Kr., Decary, F. & Engelfriet, C.P. (1977) The detection of granulocyte alloantibodies with an indirect irnmunofluorescence test. British Journal of tfaematology, 36, 533-544. Werner, G., von dem Borne, A.E.G.Kr., Bos, M.J.E., Tromp, J.F., van der Plas-van Dalen, C.M., Visser, F.J., Engelfriet, C.P. & Tetteroo, P.A.T. (1985) Localisation of the human NAI alloantigen on neutrophil Fc-y-receptors.
287
In: Leucocyte Typing II. (eds Reinherz, E.L., Haynes, B.F., Nadler, L.M. & Bernstein, I.D.), 3, 109-121. Springier-Verlag, New York. Yomtovian, R., Press, C., Engman, H., K h e , W., Clay, M. & McCullough, J. (1984) Severe pulmonary hypersensitivity associated with passive transfusion of a neutrophilspecific antibody. Lancet, i, 244-245.
Location of the granulocyte-specific antigen LAN on the Fc-receptor I11 P. Metcalfe and A. H. Waters Deparrmenr of
Haemalology. Si Barrholornew’s Hospiral and Medical College,
London E C l A 7BE. U.K. Receiced 9 January 1992; arcepred for publicarion 18 June 1992
~
This report investigates the location of the binding site of a recently described granulocytespecific alloantibody, designated LAN, which causes alloimmune neonatal neutropenia. The LAN antigen
SUMMARY.
Granulocyte-specific alloantibodies are clinically important in neonatal alloimmune neutropenia, nonhaemolytic febrile transfusion reactions (Minchinton & Waters, 1984) and transfusion-related acute lung injury (Yomtovian el al., 1984). Of the granulocytespecific alloantigens described so far (McCullough el al., 1987). only the NAI/NA2 and NBI antigens have been found to be located on particular glycoproteins (Werner e f al., 1985; Huizinga e f al., 1990a; Stroncek e f al., 1990). Recently, a new granulocyte-specific alloantibody, designated LAN, was described in an Australian Aboriginal mother of a baby with neonatal alloimmune neutropenia (Rodwell et al., 1991). This report describes the location of the binding site of the LAN antibody on the Fc-receptor 111 (FcRIII).
MATERIALS A N D METHODS LAN antibody The LAN antibody is a granulocyte-specific alloantibody which recognizes a previously undefined antigen present at high frequency ( > 99%) in predominantly Caucasian populations (Rodwell et al., 1991). The antibody is of the IgG class and reacts strongly in immunofluorescence and agglutination techniques. Monoclonal antibodies
The following mAb were used: anti-FcRIII (CLBFcRgran/l),anti-NA I (CLBgran/l I), both supCorrcspondence: Dr P. Metcalfe. Department of Haematology. SI Barrholomew’s Hospital and Medical College. West Smithfield. London EClA 7BE. U . K .
is shown to be situated on the Fc-receptor I11 (FcRIII). Key words: alloimmune neonatal neutropenia, FcRIII, granulocyte alloantibody, LAN antibody.
plied by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, and anti-Gp IIb/IIIa (NIB 85/661), supplied by The National Institute for Biological Standards and Control, Potters Bar. U.K.
Granulocyte separafion andfixation
Granulocytes were isolated from freshly collected, EDTA-anticoagulated, peripheral blood (1 volume 5 % Na2 EDTA:9 volumes blood). Two millilitres of dextran solution [5% dextran, Sigma, average mot. wt 162.000, in phosphate-buffered saline (PBS)] were mixed with each 10 ml of blood. After incubation at 37°C for 30 min the leucocyte-rich supernatant was removed and 2 ml were underlayered with 2 ml lymphocyte separation medium (LSM) (Ficoll-hypaque sp. gr. 1.077, Flow Laboratories) and 2 ml Monopoly resolving medium (MPRM) (Ficoll-hipaque sp. gr. 1.114, Flow Laboratories) in a 10-ml conical centrifuge tube. After centrifugation at 2,500 g for 5 min. the granulocytes were removed from the LSM/ MPRM interface and washed three times (400g, 5 min) in PBS/BSA (0.2%). The cells were either resuspended in PBS/BSA nr fixed in 1 % para-formaldehyde (5 min at room temperature) and washed twice more in PBS/ BSA and used as described below. Lymphocytes, used in preliminary studies to demonstrate the specificity of MAIGA reactions, were obtained from the leucocyte-rich supernatant/LSM interface at the same time as the granulocyte preparation and were processed in the same way as granulocytes.
284
P . Melcalfe and A . H . Waters
Granulocyte immunofluorescence tesl (GIFT)
Serum LAN and anti-FcRIII mAb were tested against granulocytes from a patient with paroxysmal nocturnal haemoglobinuria (PNH) and a normal donor, using the granulocyte immunofluorescence test (Verheugt et al., 1977). Pooled AB serum from 10 nontransfused male donors was used as a negative control and serum containing strong multispecific HLA antibodies was used as a positive control. Results were assessed by fluorescence microscopy using a scoring +) system (negative, weak, and the percentage of fluorescing cells was calculated by counting 300 cells.
+, + +, + + +, + + +
Monoclonal anribody-specijc immobilization granulocyte antigens ( M A I G A ) assay
00'
This assay (Fig. 1) was developed directly from the MAIPA assay for platelet antibodies described by Kiefel el al. (1987). Briefly, a pellet of 0.5-1.0 x lo6 granulocytes was resuspended in 30 pl human serum or plasma to be tested and incubated at 37°C for 30 rnin in a U-well microplate. After washing twice in PBS/BSA the granulocytes were resuspended in 30 pl mouse mAb, as detailed above, and incubated at 37°C for 30 min. The cells were then washed twice more in PBS/ BSA and lysed by the addition of 100 pI Tris buffered saline (TBS) containing 0.5% Nonidet P-40 and incubated at 4°C for 30 min. The granulocyte lysate was then transferred to a 2-ml conical tube and centrifuged at 11,600 g for 30 rnin at 4°C to remove
particulate matter. Sixty microlitres of the resulting supernatant were removed and diluted with 180pl TBS wash buffer (0.5%Nonidet P-40,0.05% Tween 20 and 0.5 mmol CaC12). One hundred microlitres of diluted granulocyte lysate were transferred, in duplicate to a flat-well microplate coated with goat anti-mouse IgG [prepared by overnight incubation of 100 p1 goat antimouse IgG (sera-tab, code SBA 1030-01)at 3 pg/ml in carbonate coating buffer, pH 9.6, followed by four washes and 30 rnin blocking in TBS wash buffer]. After incubation at 4°C for 90 min the microplate wells were washed four times with 200 pl TBS wash buffer followed by the addition of 100 pl alkaline phosphatase labelled anti-human IgG (Jackson, code 109-055008) diluted 1 :3000 in TBS wash buffer. After further incubation at 4°C for 90 rnin the wells were washed four times in TBS wash buffer and 100 p1 substrate solution [ 1 mg/ml p-nitrophenyl phosphate (Sigma) in diethanolamine buffer, pH 9.81 were added to each well. The resulting colour change was measured at 30 rnin using a dual wavelength photometer (Bio-Rad model 450). Results were expressed as the mean absorbance at 405 nm of duplicate tests minus the mean of eight blanks containing TBS wash buffer instead of granulocyte lysate. Pooled AB serum was used as a negative control. Anti-NA2 was used as a positive control in the MAIGA assays utilizing antiFcRIII mAb, but human NAI or NA2 antibodies could not be used as a positive control in the MAIGA assays utilizing anti-NAI mAb because of mutual inhibition. In some experiments lymphocytes were used in exactly the same way as granulocytes.
enzyme-labelled goat anll-human IgG
goat anti-mouse IgG
microplate
Fig. 1. Representation of the MAlGA assay.
Locarion of the granulocyte-specrfic antigen LAN
285
Table 1. GIFT results of anti-FcRIII
mAb and LAN antibody tested against granulocytes from a PNH patient and a normal donor, measured as a percentage
PNH patient
Antibody
Weakor negative
Anti-FcRIII mAb LAN antibody Negative control (AB serum) Positive control (anti-HLA)
73 74 100 0
RESULTS Immunojhorescence studies on PNH granulocytes Granulocytes were obtained from a 42-year-old woman who had paroxysmal nocturnal haemoglobinuria (PNH). Only 27% of the granulocytes showed strongly positive reactions when tested with antiFcRIII mAb, the remainder giving mostly weak positive or occasionally negative reactions, compared to 97% strongly positive reactions with normal donor granulocytes (Table I). The LAN antibody showed the same pattern of reactions when tested with granulocytes from the PNH patient, 26% of the cells were strongly positive and the remaining 74% of cells showed weak or negative reactions, while 98% of normal donor granulocytes were strongly positive. This weaker reaction pattern seen with PNH granulocytes was not seen in over 600 normal donors tested with the LAN antibody (Rodwell et al., 1991). These reactions suggest that the binding site of the LAN antibody is located on a membrane protein which is deficient in PNH cells. MAIGA studies The specificity of the MAIGA assay was shown by reactions of the LAN antibody with granulocytes and lymphocytes in tests using three different mAbs (Table 2). Positive reactions were obtained only when Table 2. Specificity of MAIGA assay
Immobilizing mAb Antibody
Cells
IIb/IIIa
FcRIlI
NAI
LAN LAN
Lymphocytes Granulocytes
0.020* 0.029
0.065 0.557
0.02 I 0.566
Values are mean absorbance of duplicate tests on each of donors.
IWO
Normal donor
+ + + or
++++ 27 26 0 100
Weakor negative 3 2 100 0
+ + + or ++++ 97 98 0 100
the LAN serum was tested against granulocytes in combination with either anti-FcRIII or anti-NA1 mAbs. All reactions against lymphocytes and reactions against granulocytes in combination with an irrelevant monoclonal antibody (anti-GP IIb/IIIa) were negative. The LAN alloantibody was tested against PFAfixed and unfixed granulocytes using both anti-FcRIII and anti-NAI mAb to immobilize the FcRIII receptor in the MAIGA. Positive results were obtained with both mAb (Fig. 2a and b), indicating that the LAN antigen is on the FcRIII receptor. Differentiation between positive and negative results was greater when PFA-fixed granulocytes were used. DISCUSSION
In contrast to platelet and red cell antigens, little is known about the location of the antigens recognized by granulocyte alloantibodies. The neutrophil-specific alloantigens NAI and NA2 have been localized on FcRIII (Werner et al., 1985; Huizinga et al., 1990a) and the NBI antigen has been demonstrated on a 5864 kDa glycoprotein present on the neutrophil membrane and in secondary granules (Stroncek et al., 1990). This report demonstrates that the LAN antigen is situated on FcRIII. This was suspected after immunofluorescence studies on PNH granulocytes showed that the LAN antibody gave the same reaction pattern as anti-FcRIII mAb. The reactions with PNH granulocytes were typical of those seen by van der Schoot et al. (1990) when they described the reactions of granulocytes from 20 patients with PNH tested in a cytofluorimetric assay with antibodies against glycosyl-phosphatidylinositol (GP1)-linked membrane proteins, including FcRIII. An antigen capture assay (MAIGA) was developed to study this further. FcRIII was immobilized in microplates by anti-FcRIII and -NAI mAbs and binding of the LAN antibody to FcRIII was demonstrated by ELISA. PFA-fixation of granulocytes resulted in better differentiation between positive and
P . Metcalfe and A . H . Waters
T, 0.5
1
that the LAN antibody was an iso-antibody against FcRIII formed in an “A-null’ mother (Huizinga et al., 1990b) was excluded by re-testing the mother and showing that her neutrophils were NA 1/NA2 positive (R. Minchinton, personal communication).
ACKNOWLEDGEMENTS We wish to thank Dr R. Rodwell for providing the LAN antibody, Dr R. Minchinton for typing the LAN neutrophils and Dr J. A. L. Amess who provided blood samples from the patient with PNH. Unfixed granulocytes
PFA.fixed granulocytes
REFERENCES
0.4
0.3
02
0.1
00
Unfixed granuiaytes
PFA-fixed granulocytes
Fig. 2. Reactions of the LAN antibody with PFA-fixed and unfixed donor granulocytes in the MAIGA assay, using, (a) anti-FcRIII mAb and granulocytes from four NA2 positive donors, (b) anti-NAl mAb and granulocytes from three NAI positive donors. Each column represents the mean of duplicate measurements on four donors and three donors, respectively, and the error bars indicate 1 standard deviation from the mean. (M)Negative control, (R)LAN antibody, (0)positive control.
negative results, as also shown for lymphocytes in the monoclonal antibody-specific immobilization of leucocyte antigens (MAILA) assay (Mueller-Eckhardt er al., 1989). Now that many anti-granulocyte mAb are available, the MAIGA technique may be useful in the investigation of other granulocyte allo- and autoantibodies. The LAN antigen is the second granulocyte-specific alloantigen to be located on FcRIII. The possibility
Huizinga, T.W.J., Kleijer, M., Tetteroo, P.A.T., Roos, D. & von dem Borne, A.E.G.Kr. (l990a) Biallelic neutrophil NA-antigen system is associated with a polymorphism on the phospho-inositol-linked Fcy receptor 111 (CD16). Blood, 75,213-217. Huizinga, T.W.J., Kuijpers, R.W.A.M., Kleijer, M., Schulpen, T.W.J., Cuypers, H.T.M., Roos, D. & von dem Borne, A.E.G.Kr. (1 990b) Maternal genomic neutrophil FcRIII deficiency leading to neonatal isoimmune neutropenia. Blood, 76, 1927-1932. Kiefel, V., Santoso, S., Weisheit, M. & Mueller-Eckhardt, C. (1987) Monoclonal antibody specific immobilisation of platelet Antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies. Blood, 70, 1722-1726. McCullough, J., Clay, M. & Kline, W. (1987) Granulocyte antigens and antibodies. Transfusion Medicine Reviews, 1, 150- 160. Minchinton, R.M. & Waters, A.H. (1984) The Occurrence and significance of neutrophil antibodies. British Journal of Haematology, 56, 521-528. Mueller-Eckhardt, G.,Kiefel, V., Schmidt, A., llusty, A., Santoso. S. & Mueller-Eckhardt, C. (1989) Discrimination of antibodies against antigens of different M H C loci in human sera by monoclonal antibody-specific immobilisation of leukocyte antigens. Human Immunology, 25, 125134. Rodwell, R.L., Tudhope, D.I., ORegan, P.O., Minchinton, R. &Waters, A.H. (1991) Alloimmune neonatal neutropenia in Australian aboriginals: an unrecognised disorder? Tramfusion Medicine, 1, 63-67. Stroncek, D.F., Skubitz, K.M. & McCullough, J.J. (1990) Biochemical characterization of the neutrophil-specific antigen NBI. Blood, 75, 744-755. van der Schoot, C.E., Huizinga, T.W.J., van’t Veer-Korthof, E.T., Wijmans, R., Pinkster, J. & von dem Borne, A.E.G. Kr. ( 1 990) Deficiency of glycosyl-phosphatidylinositollinked membrane glycoproteins of leucocytes in paroxysmal nocturnal haemoglobinuria, description of a new diagnostic cytofluorometric assay. Blood, 76, 1853-1 859.
Location of the granulocyte-specific antigen L A N Verheught, F.W.A., vondem Borne, A.E.G. Kr., Decary, F. & Engelfriet, C.P. (1977) The detection of granulocyte alloantibodies with an indirect irnmunofluorescence test. British Journal of tfaematology, 36, 533-544. Werner, G., von dem Borne, A.E.G.Kr., Bos, M.J.E., Tromp, J.F., van der Plas-van Dalen, C.M., Visser, F.J., Engelfriet, C.P. & Tetteroo, P.A.T. (1985) Localisation of the human NAI alloantigen on neutrophil Fc-y-receptors.
287
In: Leucocyte Typing II. (eds Reinherz, E.L., Haynes, B.F., Nadler, L.M. & Bernstein, I.D.), 3, 109-121. Springier-Verlag, New York. Yomtovian, R., Press, C., Engman, H., K h e , W., Clay, M. & McCullough, J. (1984) Severe pulmonary hypersensitivity associated with passive transfusion of a neutrophilspecific antibody. Lancet, i, 244-245.
