British Iournal of Haematologu, 1992. 81,552-557
HLA-DR expression by platelets in acute idiopathic thrombocytopenic purpura LYNN K. BOSHKOV,J O H N G . KELTON A N D PHILIPF. HALLORAN Departments of Medicine and Pathology, McMaster University Medical Centre, Hamilton, Ontario, Department of Medicine, University of Alberta, Edmonton, Alberta, and Canadian Red Cross Blood Transfusion Service, Edmonton Centre, Edmonton, Alberta
Received 27 September 7 991; accepted for publication 18 March 1992
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Summary. Induction of expression of MHC class II antigens on the surface of cells that do not ordinarily express these proteins has been implicated in the pathogenesis of autoimmunity in diabetes mellitus and autoimmune thyroiditis. Platelets express class I but not class I1 HLA antigens. In this report, we describe a child with acute idiopathic thrombocytopenic purpura who at the time of the thrombocytopenic episode had class II (HLA-DR) antigens on his platelets. Following recovery, the HLA-DR antigens were no longer present on the platelets. We postulated that class I1 had been induced on his megakaryocytes by a cytokine such as
interferon gamma, and that the induced expression of class I1 antigens contributed to the autoimmune disorder. To substantiate this possibility we next studied class I and II antigen expression on an erythroleukaemia cell line (HEL), which has many megakaryocytic features. Following treatment of HEL cells with interferon gamma, class I expression was increased and HLA-DR antigens were induced. These observations suggest that cytokine-mediated induced HLA-DR expression may contribute to the pathogenesis of a subset of thrombocytopenias.
Class I and class I1 MHC glycoproteins function physiologically to present antigens on the cell surface for recognition by T lymphocytes (recently reviewed by Yewdell & Bennink, 1990). Antigen presented in the context of class I MHC molecules is recognized by CD8 (suppressor-cytotoxiccells) and stimulates secretion of interleukins and other cytokines by T cells. Antigen presented in the context of class I1 MHC molecules is recognized by CD4 (helper) cells initiating the production of antibody by B cells. Almost all nucleated cells in the body express class I antigens: however, class I1 expression is much more restricted and found primarily on activated macrophages, B cells, activated T cells and dendritic cells. In addition, class I1 can be induced on many other cells by interferon gamma (Basham & Merigan. 1983: Collins et al, 1984: Wong et al, 1984: Fiers et al. 1985: Niederwieser et al, 1988). The induction of class tI expression on cells not normally carrying these antigens has been postulated to lead to inappropriate presentation of self or altered self antigens with subsequent T-helper cell recognition and autoantibody production (Bottazzo et al, 1983: Editorial, 1985). Such induced expression of class II antigens has been demon-
strated on thyroid cells and pancreatic islet cells, raising the possibilitythat altered expression of membrane proteins plays a role in the pathogenesis of autoimmune thyroiditis and diabetes mellitus (Hanafusa et al. 1983: Bottazzo et al, 1985: Pujol-Borrell et al, 1986, 1987; Grubeck-Loebenstein et al. 1988). In humans, haematopoietic progenitor cells, but not their mature progeny, express HLA-DR antigens (Fitchen et al, 1981:Fakenbergetal,1984;Amatrudaetal, 1987:Buschet al, 1987). Interferon gamma can induce HLA-DR expression on normal and leukaemic human myeloid cells and on myeloid cell lines (Basham et al, 1984: Virelizier et al, 1984: Koeffler et al, 1984; Amatruda et al, 1987). In the megakaryocyte Lineage the colony-forming unit-megakaryocyte (CFU-MK) appears to express HLA-DR whereas the burstforming unit-megakaryocyte (BFU-MK) does not (Briddell et al, 1989). Mature platelets express HLA class I antigens but not HLA class I1 antigens (Rabellinoet al, 1979; Klein, 1986: Kunicki. 1988). Loss of HLA-DR by megakaryocyte precursors may occur before the gain of the glycoprotein IIb/IJIa antigen (Koike et al. 1987; San Miguel et al. 1987). We hypothesized that transient induction of class II antigens on megakaryocytes by cytokines such as interferon gamma (perhaps stimulated by a viral infection) might underlie certain acute immune thrombocytopenias.
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Correspondence:Dr L. K. Boshkov, Room 4B2.16.Walter Mackenzie Centre, University Hospital, 8440 112 Street, Edmonton. Alberta, Canada T6G 2B7.
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HLA-DR Expression by Platelets in Acute ITP In this report we describe a child with an antecedant upper respiratory infection and a peripheral smear compatible with viral infection who presents with acute idiopathic thrombocytopenic purpura (ITP). Class 11-HLA-DK antigens were present on the child’splatelets at the time of the thrombocytopenia. The expression of these antigens was lost as the child recovered. We propose that induced expression of class I1 antigens on platelets might be involved in the pathogenesis of the immune thrombocytopenic disorder in this child. In vitro studies of the cell line HEL, an erythroleukaemia line with megakaryocytic features, demonstrated that these cells can express class I1 antigens following stimulation with interferon gamma. CASE REPORT A 4-year-old boy presented with a 1-d history of petechiae
and bruising following a respiratory tract infection approximately 10 d prior to admission. Physical examination demonstrated a diffuse petechial rash and generalized purpura without lymphadenopathy or splenomegaly. The platelet count was 5 x 109/1, white count was 4 x 10y/l with granulocytopenia (granulocytes 1 .O x 1 0 y / l ) and reactive lymphocytes. The haemoglobin was 12.7 g/dl. The direct antiglobulin test and antinuclear antibody were negative. C3, C4 and quantitative immunoglobulins were normal. The bone marrow demonstrated increased numbers of megakaryocytes. The patient was treated with 4 mg/kg of prednisone per day and the platelet count rose to normal over the next week and a half. An older sister of this child had autoimmune haemolytic anaemia and immune thrombocytopenia at age 4 months that was treated and cured by splenectomy at age 2. MATEKIALS A N D METHODS Monoclonal antibodies used in platelet and cell culture studies. Antiplatelet antibodies against GP Ilb/lIIa (Kaj-1) and GP Ib/ IX (Beb-1) were produced as described (Horsewood et al, 1991). Monoclonal antibody against the platelet Fc receptor (IV.3)(Kosenfeld et al, 1985 ) was a gift of Dr Clark Anderson, Ohio State University Columbus, Ohio. Antibodies were fluorescein isothiocyanate (FITC) conjugated to fluorescein/ protein ratios of 7.4 (anti-IIb/IIIa). 2.4 (anti-Ib/IX) and 6.1 (anti-Fc receptor). Phycoerythrin (PE)-conjugated anti-HLADR (I3),anti-CD4 (T4).and isotype IgG2a control (MS IgG2a) were obtained from Coulter Immunology, Burlington. Ontario, Canada. 13 is a nonpolymorphic class I1 (HLA-DR)Ia antigen of 34.29 kD molecular weight (Toddet ul, 1984).PEconjugated anti-HLA-DR (IOT2a)was obtained from AMAC/ Bio/Can, Mississauga, Ontario. IOT2a reacts with a monomorphic class I1 epitope (Kebai et al. 1983). Monoclonal antibody against a monomorphic determinant on HLA-A,B.C (W6/32) was obtained as a hybridoma from American Type Culture Collection (Rockville. Md.) and purified using Staph. protein A. Monoclonal antibody IgG2a isotype control (UPC 10) was obtained from Sigma, St Louis, Mo. Unconjugated murine monoclonal antibodies were visualized by the addition of FITC-conjugated F(ab)’2 adsorbed goat anti-mouse antibody (Cappel. West Chester, Pa.). Flow cytometric analysis oJ’platelets. (1) Patient and control
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platelets. Flow cytometric analysis was performed using platelets from the child collected on days 3. 8 and 21 following initiation of corticosteroid therapy, when the platelet count was 19. 42 and 1 6 7 x 109/1respectively. Platelets from a normal adult control were processed in parallel and adjusted to platelet count comparable to patient platelets with autologous platelet poor plasma before antibody binding. Additional control platelets from an adult with chronic ITP (platelet count 5 5 x 10y/l) were studied in parallel with the patient’s day 8 platelets. Other subjects who were studied included 25 normal children, six normal adults, four children with chronic ITP, seven adults with chronic ITP, and one child and one adult with acute ITP. ( 2 ) Antibody binding. Whole blood was collected into EDTA, allowed to settle at 1 g for 12 h. and the platelet rich plasma aspirated. Aspirated plasma had less than 0.8%white blood cells. Antibody binding was done directly in platelet rich plasma with 3-5 x lo6 platelets in a total reaction volume of 100 pl. The platelets were mixed separately with the FITC-conjugated monoclonal antibodies against GP IIb/ IIIa (Raj-l),GP Ib/IX (Beb-l), the platelet Fc receptor (IV.3), and with the PE-conjugated monoclonal antibodies against HLA-DR (13)and CD4 (T4).The monoclonal antibodies were used at supersaturating concentrations. Anti-HLA-DR and anti-CD4 do not normally bind to platelets and were added in quantities recommended by the manufacturer as sufficient to saturate 1 x 10‘ test white blood cells. Both a FITC-conjugated antiplatelet antibody and a PE-conjugated antibody were added simultaneously to some samples. Antibodies were allowed to bind to platelets for 1 h at 22OC. and then 1 x lo6 platelet aliquots (20-50 PI) were diluted in 1 ml of filtered calcium-albumin-free Tyrode’s buffer ( 137 mmol/l NaCI. 12 mmol/l NaHC03, 2.6 mmol/l KCI. 2 mmol/l MgCI2. 5.6 mmol/l glucose, pH 7.4) and 1 5 000 events analysed using a Coulter EPICS V flow cytometer. Platelets were identified by their forward and side light scatter profiles. Gates were set to exclude red cells and white cells. Quadrant analysis (green fluorescence on the abscissa and red fluorescence on the ordinate) was performed using Coulter software on platelet samples stained with both FITC- and PE-conjugated antibodies. H E L cell cultures and interferon gamma induction. HEL. a human erythroleukaemia cell line with megakaryocytic features (Martin & Papayannopoulou. 1982; Breton-Gorius & Vainchenker, 1986) was obtained from American Type Culture Collection (Rockville, Md.). Cells were cultured in KPMI 1640 medium with 10%heat-inactivated fetal bovine serum (Gibco Canada, Burlington. Ontario) and maintained in exponential growth. Human recombinant interferon gamma (Amersham, Oakville. Ontario, Canada) was added at the time of cell passage (initial cell concentration 1 x lo5 cells/ml) in doses ranging from 0 to 500 units/ml. After 3 d the cells were harvested, washed twice, and resuspended in calcium-albumin-free Tyrode’s buffer. Trypan blue staining confirmed > 90% viability before flow cytometry studies. Aliquots of 5 x lo5HEL cells in a total reaction volume of 200 pI were mixed separately with predetermined saturating concentrations of FITC-conjugated anti-GP Ib/D( (Beb-1).
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L. K. Boshkov, J. G. Kelton and P. F . Halloran
LOG GREEN FLUORESCENCE (FITC ANTIGCIUIX)
LOG FLUORESCENCE (FITC ANTIGCIWIX)
4 4 LOG GREEN FLUORESCENCE EFlTC ANTIGCIMXI
Fig 1. Quadrant analysis of the staining of patient day 8 platelets and platelets of a normal adult control run in parallel. Green fluorescence representing staining with the FITC-conjugated platelet-specific anti-GP Ib/M monoclonal antibody (Beb-1) is on the abscissa and red fluorescencerepresenting staining with the PE-conjugated anti-DR monoclonal antibody (13) or the PE-conjugated anti-T4 control antibody is on the ordinate. (a) Patient day 8 platelets stained with FITC anti-GP Ib/IX and PE anti-DR. (b) Patient day 8 platelets stained with FITC anti-GP Ib/rX and PE anti-T4. (c) Normal adult platelets stained with FITC anti-GP Ib/IX and PE anti-DR. A subpopulation of patient platelets showed unique positivity for the DR antigen.
anti-GP IIb/IIIa (Raj-1). and anti-platelet Fc receptor (IV.3) and with PE-conjugated anti-HLA-DR antibodies (I3 and IOTZa), anti-CD4 (T4) and isotype IgG2a control (MS IgG2a). Unconjugated anti-HLA-A,B,C (W6/32) and isotype IgG2a (UPC 10) were also mixed with HEL cells, followed by FITC F(ab)’, goat anti-mouse antibody (added 15 min after the primary antibody). Antibodies were allowed to bind for 30 min on ice. Samples were washed with 10 volumes of buffer, resuspended in 400 p1 of buffer, and 5000 events analysed per sample using a FACScan flow cytometer (Becton-Dickinson Immunocytometry Systems, Mountainview, Calif.). The results were expressed as histograms of log cell fluorescence intensity on the abscissa and cell number on the ordinate. RESULTS Platelet studies Patient day 3, 8 and 2 1 platelets stained strongly positive
with platelet-specific monoclonal antibodies against GP IIb/ m a and GP Ib/IX. A subpopulation of day 3 and 8 patient platelets were also strongly positive for HLA-DR antigen using the monoclonal antibody I3 (Fig l a , Table I). Patient platelets did not stain with PE anti-T4 (Fig lb) although this antibody stained lymphocytes appropriately. Quadrant analysis c o n b e d that the patient’s HLA-DR positive particles (platelets) were also positive for platelet-specific monoclonal antibodies (Fig l a ) excluding contamination by non-platelet particles (Ault, 1988). Normal adult platelets and platelets from an adult patient with chronic ITP tested in parallel with patient day 8 platelets showed positive staining with the monoclonal antibody against GP Ib/IX, but did not react with the monoclonal antibody against HLA-DR (Table I, Fig lc). Patient and control platelets demonstrated similar staining with the FITC-conjugated monoclonal antibody against the platelet Fc receptor (IV.3).As the child recovered,
HLA-DR Expression by Platelets in Acute ITP Table 1. Platelet positivity for anti-DR antibody staining in patient and controls.
% gated platelets Platelet count x 10'/1 mean (range)
n
positive for FITC-anti GP Ib/IX and PE-anti DR mean (range)
Patient Day 3 Day 8 Day 21
19 42 167
Adult chronic ITpt Normal adultt
55 214
1.13 0.26 0.55 (0-2.48) 0.25 (0-1.0)
39.4' 22.8 1.8
Normals Children (age 1-9) Adults
25 6
322 (250-463) 271 (214-350)
Chronic ITP Children (age 5-1 3 ) Adults
4 7
58 (1 1-109) 57 (6-1 32)
0.99 (0.04-1.70) 1.02 (0.04-2'64)
Acute ITP Children (age 3) Adults
1 1
12 10
4.16 3.88
Day 3 patient platelets were stained with FITC-anti GP IIb/IIIa and PEanti DR. t Run in parallel with patient day 8 platelets.
the percentage of platelets carrying the HLA-DR antigen progressively declined (Table I). Identically processed platelets from a further 2 5 healthy pediatric controls, six normal adult controls, four pediatric patients with chronic ITP. and seven adult patients with chronic ITP were tested. All platelets showed strongly positive staining with the anti-GP Ib/IX monoclonal antibody
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but none was significantly positive for HLA-DR antigen. Two additional thrombocytopenic patients with acute R P were tested, one pediatric and one adult. Both were negative for HLA-DR antigen (Table I).
Induction ofclass I and I1 on HEL cells by gamma interferon HEL cells, like platelets, stained strongly positive using antiGP Ib/IX (Beb-1). anti-GP IIb/IIIa (Raj-1). and anti-platelet Fc receptor (IV.3) antibodies. HEL cells also stained strongly positively with the anti-class I antibody (W6/32). Uninduced HEL cells stained only weakly with the PE-conjugated antiHLA-DR monoclonals (I3 and IOT2a) when compared with a PE-isotype IgG2a control (MS IgG2a). Anti-CD4 (T4)was not reactive. Following treatment with interferon gamma, class I surface antigen expression increased as shown by increased W6/32 binding (Fig 2a). Induction of surface HLA-DR antigen also occurred (Fig 2b) as shown by comparable increases in binding of both anti-HLA-DR antibodies. Induction of both class I and class I1 HLA-DR showed a dose response and was maximal at an interferon concentration of 200 pg/ml. The interferon gamma stimulation experiments were performed on two separate occasions with the same results. Binding of anti-GP Ib/IX, anti-GP IIb/IIIa. and isotype controls was not changed following interferon treatment (data not shown). Cell viability as assessed by trypan blue exclusion was not affected by interferon treatment, nor did interferon treatment cause appreciable morphological changes as assessed visually or by changes in forward and side scatter parameters flow cytometrographically. DISCUSSION Idiopathic thrombocytopenic purpura (ITP) is a common autoimmune disease with two general patterns of presentation and chronicity. Most adults with ITP have chronic disease with an insidious onset, whereas young children tend to have an acute onset of self-limited thrombocytopenia which often is preceded by a viral infection. Although the
f 2QB
4Q0
CQP
OPQ
RED FLUORESCENCE Fig 2. Response of HEL cells to interferon gamma (200 pg/ml):-, untreated: ....,IFN 200. (a)Interferon gamma increases surface expression of HLA class I antigen as shown by increased binding of the anti-HLA-A.B,Cmonoclonal antibody W6/32 visualized by FITC-conjugated F(ab)'2 goat anti-mouse antibody. (b) Interferon gamma induces surface expression of HLA-DR as shown by increased binding of PE-conjugated monoclonal antibody 13.
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L. K. Boshkov, 1. G. Kelton and P. F. Halloran
target antigens of ITP have been identified as platelet glycoproteins (most often glycoprotein IIb/IIIa) (Karpatkin, 19 8 5). the underlying pathogenesis of autoantibody formation remains uncertain and may be heterogeneous. We postulated that transient induced class I1 expression by platelets might explain certain acute immune thrombocytopenias and perhaps be the initiating event in chronic antibody-mediated thrombocytopenia in patients unable to limit autoantibody formation. Using flow cytometric analysis with monoclonal antibodies, we found that class I1 DR antigens were not normally present on platelets from normal adults and children, nor were they present on the platelets from a number of adults and children with chronic ITP.However, the child described here had an acute episode of ITP in which HLA-DR antigens were detected on the surface of the platelets during the acute thrombocytopenic episode. As the child recovered, the proportion of platelets carrying antigens declined to progressively lower numbers. We were careful to determine that the HLA-DR antigen expression on the child’s platelets was not due to technical factors such as contaminating white cells. the non-specific adsorption of antibody molecules, or the binding of monoclonal antibody to platelet Fc receptors. Unfortunately we have only been able to test one additional thrornbocytopenic child with acute ITP. This second child’s platelets were negative for HLA-DR (Table I). Thus the incidence of HLA-DR positivity on platelets in acute childhood ITP is unclear, and it is possible our patient is unique. To demonstrate the feasibility of induction of HLA-DR expression on megakaryocytes. we studied HLA antigen expression on the HEL erythroleukaemia cell line which has megakaryocytic features. Analysis of HEL cells demonstrated that class I antigens were present in large numbers on the cell surface. In the uninduced state there was little class I1 expression on the HEL cells. However, following the addition of interferon gamma, class I expression was increased and class I1 HLA-DR expression was induced. Induction of HLADR on HEL cells by interferon gamma was also noted by Amatruda et a1 (1987).Very recently increased class I mRNA expression and induction of HLA class I1 mRNA expression by interferon gamma was reported on another human megakaryocytic cell line, Dami (Monte et al. 1991). HLA-DR is expressed on early megakaryocyte progenitors (CFU-MK) (Briddell et al, 1989) but is lost subsequently (Koike et al, 1987: San Miguel et al, 1987). Although the role of class I1 products in antigen presentation is well established, their role in megakaryopoiesis is unclear. Class I1 expression by mature platelets has not been previously reported. While the transient expression of HLA-DR antigens on the platelets from this child with acute ITP could have been coincidental, it is our hypothesis that in this case (and perhaps others) an acute viral infection or other immunological insult generated cytokines including interferon gamma. These cytokines cause the megakaryocytes/platelets to express class I1 antigens resulting in the inappropriate presentation of platelet-specific protein to the immune system with subsequent autoantibody production and antibodymediated destruction of platelets. Recovery from the infection results in the loss of the class I1 antigen from the surface of the
platelets with resolution of the immune thrombocytopenia. For the present, the possible role of megakaryocytes as antigen-presenting cells, the extent and in vivo significanceof induction of class I1 antigens on megakaryocytes by cytokines, and the significance of platelet HLA-DR expression remain speculative. ACKNOWLEDGMENTS We thank Barbara Kurc Bagnarol for performing the quadrant analysis of platelet samples on the Coulter EPICS V and Peter Horsewood for FITC-conjugation of the anti-platelet antibodies. These studies were supported in part by a grant from the Medical Research Council of Canada and by grant 71 76 from the Special Services and Research Development Fund of the University of Alberta to L.K.B. This work was presented in part at the American Society of Hematology Meeting in Denver, Colorado, December 1991 (Boshkov et al, 1991). REFERENCES Amatruda, T.T., Bohman. R., Ranyard. j. & Koeffler. H.P. (1987) Pattern of expression of HLA-DR and HLA-DQ antigens and mRNA in myeloid differentiation. Blood. 69, 1225-1236. Ault. K.A. (1988) Flow cytometric measurement of platelet-associated immunoglobulin.Pathology and Immunopathology Research. 7, 395-408. Basham. T.Y. & Merigan. T.C. (1983) Recombinant interferongamma increases HLA-DR synthesis and expression. journal of Immunology. 130, 1492-1494. Basham. T.. Smith. W.. Lanier, L.. Morhenn. W. & Merigan. T.T. (1 984) Regulation of expression of class 11 major histocompatibility antigens on human peripheral blood monocytes and Langerhans cells by interferon. Human Immunologu. 10, 83-93. Boshkov, L.K.. Kelton. J.G. & Halloran. P.F. (1991) HLA-DR expression by platelets in acute thrombocytopenia.Blood, 78, (10). Suppl. 1, Abstract 574. Bottazzo, G.F., Dean. B.M., McNally. J.M.. MacKay, E.H., Swift, P.G.F. & Gamble, D.R. (1 985) In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulinitis. New England journal of Medicine, 3 13, 3 5 3360. Bottazz~.G.F..Pujol-Borrell,R.. Hanafusa. T. & Feldmann. M. (1983) Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity. Lancet, ii, 1 1 1 5-1 119. Breton-Gorius. J. 81 Vainchenker, W. (1986) Expression of platelet proteins during the in witro and in vivo differentiationof megakaryocytes and morphological aspects of their maturation. Seminars in Hematology. 23, 43-67. Briddell, R.A.. Brandt, J.E., Straneva. J.E.. Srour. E.F. & H o h a n , R. (1989) Characterizationof the human burst-forming unit-megakaryocyte. Blood, 74,145-151. Busch, F.W.. Langer, M.. Pawelec. G., Ziegler. A., Wernet, P., Buhring. H.J., Meyer, P. & Muller. C. (1987)HLA-class I1 antigens on human haematopoietic progenitors. Blut. 54, 179-1 88. Collins, T.. Korman, A S . . Wake, C.T..Boss, J.M.. Kappes, D.J..Fiers. W.. Auk K.A.. Gimbron, M.A., Jr, Strominger, J.L. & Pober. J.S. (1984) Immune interferon activates multiple class I1 histocompatibility complex genes and the associated invariant chain in human endothelial cells and dermal fibroblasts. Proceedings o/ the National Acudemu of Sciences of the United States of America, 81, 491 7 4 9 2 1 .
HLA-DR Expression by Platelets in Acute 1TP Editorial (1985) What triggers autoimmunity? Lancet, ii, 78-79. Falkenberg. J.H.F.. Jansen. J., van der Vaart-Duinkerken. N.. Veenhof, W.F.J.. Blotkamp. J., Goselink. H.M.. Parlevliet. J. & van Rood, J.J. (1984)Polymorphic and monomorphic HLA-DR determinants on human hematopoietic progenitor cells. Blood, 63,1125-1 132. Fiers. W.. Endler. B.. Reske, K.. Wekerle. H. & Fontana. A. (1985) Astrocytes as antigen-presenting cells. 1. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation. Journal oflmmunology, 134, 37853793. Fitchen. J.H.. LeFevre. C.. Ferrone. S. & Cline, M.J.C. (1981) Expressionof la-like and HLA-A.B antigens on human multipotential hematopoietic progenitor cells. Blood. 59, 188-190. Grubeck-Loebenstein. B.. Lonei. M.. Greenall, C.. Pirich, K.. Kassal, H., Waldhausi. W. & Feldmann. M. ( 1 988) Pathogenic relevance of HLA class II expressing thyroid follicular cells in nontoxic goitre and in Grave's disease. Journal o/ Clinical Investigation, 81, 1608-1 614. Hanafusa. T.. Chiovato. L.. Doniach. D.. Pujol-Borrell. R.. Russell, R.C.G. & Bottaao, G.F. (1983) Aberrant expression of HLA-DR antigen on thyrocytes in Grave's disease: relevance for autoimmunity. Lancet. ii, 1 1 1 1 - 1 1 1 5. Horsewood. P.. Hayward. C.P.M.. Warkentin, T.E. & Kelton, J.G. (199 1) Investigation of the mechanisms of monoclonal antibodyinduced platelet activation. Blood. 78. 101 9-1026. Karpatkin, S. (1985) Autoimmune thrombocytopenic purpura. Seminars in Hematology. 22, 260-288. Klein. J. (1986) Natural History of the Major Histocompatibility Complex, pp. 160-161. John Wiley & Sons, New York. Koeffler, H.P.. Ranyard, J., Yelton. L., Billing. R. & Bohman, R. (1984) Gamma-interferon induces expression of the HLA-D antigens on normal and leukemic human myeloid cells. Proceedings of the National Academy of Science of the United States oJ America. 81, 4080-4084. Koike, T., Aoki. S.. Maruyama, S.. Narita. M.. Ishizuka. T.. Imanaka. H.. Adachi. T.. Maeda. H. & Shibata. A. (1987) Cell surface phenotyping of megakaryoblasts. Blood. 69, 9 57-960. Kunicki, T.J. (1988) Antigens and immune receptors. Platelet Membrane Receptors: Molecular Biology, Immunology. Biochemistry and Pathology (ed. by G. A. Jamieson), p. 108. Alan R. Liss. New York. Martin, P. & Papayannopoulou. T. (1982) HEL cells: a new human erythroleukemia line with spontaneous and induced globin gene expression. Science, 21 6, 1233-1 23 5. Monte, D.. Wietzerbin. J.. Pancere. V.. Merlin, G.. Greenberg, S.M.. Kusnien, 1-P.,Capron. A. & Auriault. C. (1991 ) Identification and characterization of a functional receptor for interferon-gamma on a megakaryocytic cell line. Blood. 78, 2062-2069.
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Niederwieser, D., Auboeck. J.. Trippmair. J., Herold. M., Schuler, G.. Boeck, G.. Lotz. J., Fritsh, P. 81Huber. C. (1988) IFN-mediated induction of MHC antigen expression on keratinocytes and its influence on in vitro alloimmune responses. Journal of Immunology, 140, 2556-2564. Pujol-Borrell. R.. Todd. I., Doshi, M.. Bottazzo. G.F.. Sutton, R.. Gray, D.. Adolf. G.R.& Feldmann. M. (1987) HLA class I1 induction in human islet cells by interferon plus tumour necrosis factor or lymphotoxin. Nature, 326, 304-306. Pujol-Borrell, R.. Todd, I.. Londei, M.. Foulis. A., Feldmann, M. & Bottazzo, G.F. ( 1986) Inappropriate major histocompatibility complex class I1 expression by thyroid follicular cells in thyroid autoimmune disease and by pancreatic beta cells in type I diabetes. Molecular Biology and Medicine, 3, 159-165. Rabellino. E.M., Nachman. R.L., Williams, N., Winchester, R.J. & Ross. G.D. (1979) Human megakaryocytes. I. Characterization of the membrane and cytoplasmic components of isolated marrow megakaryocytes. Journal of Experimental Medicine, 149, 12 731287. Rebai. N.. Malissen. B.. Pierres. M., Accola, R.S.. Corte, G. & Mawas, C. (1983) Distinct HLA-DR epitopes and distinct families of HLADR molecules defined by 1 5 monoclonal antibodies (mAb) either anti-DR or allo-anti-lak cross-reacting with human DR molecule. European Journal of Immunology. 13, 106-1 11. Rosenfeld. S..I..Looney. R.J.. Leddy. J.P., Phipps. D.C.. Abraham, G.N. & Anderson, C.L. ( 1 985) Human platelet Fc receptor for immunoglobulin G. Identification as a 40,000 molecular weight membrane protein shared by monocytes. lournal of Clinical Investigation, 76,2317-2322. San Miguel. J.F., Gonzalez. M.. Orfao, A., Ojeda. E. & Canizo. M.D. (1987) Expression of immunologic markers in megakaryoblasts. Blood. 70, 1227-1228. Todd, R.F.. Meuer. S.C., Romain. P.L. & Schlossman. S.F. (1984) A monoclonal antibody that blocks class I1 histocompatibilityrelated immune interactions. Human Immunologu. 10, 23-40. Virelizier. 1.-L.. Perez, N.. Arazena-Seisdedos. F. & Dovos. R. (1984) Pure interferon gamma enhances class I1 HLA antigens on human monocyte lines. European Journal of Immunology. 14, 106-108. Wong. G.H.W., Clarke-Lewis, 1.. Harris, A.W. & Schrader. J.W. (1984) EfFect of cloned interferon-gamma on expression of H-2 and Ia antigens on cell lines of hemopoietic. lymphoid, epithelial. fibroblastic and neuronal origin. European /ournal oJ Immunologu, 14, 52-56. Yewdell, J.W. & Bennink. J.R. (1990) The binary logic of antigen processing and presentation to T cells. Cell, 62, 203-206.
HLA-DR expression by platelets in acute idiopathic thrombocytopenic purpura LYNN K. BOSHKOV,J O H N G . KELTON A N D PHILIPF. HALLORAN Departments of Medicine and Pathology, McMaster University Medical Centre, Hamilton, Ontario, Department of Medicine, University of Alberta, Edmonton, Alberta, and Canadian Red Cross Blood Transfusion Service, Edmonton Centre, Edmonton, Alberta
Received 27 September 7 991; accepted for publication 18 March 1992
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Summary. Induction of expression of MHC class II antigens on the surface of cells that do not ordinarily express these proteins has been implicated in the pathogenesis of autoimmunity in diabetes mellitus and autoimmune thyroiditis. Platelets express class I but not class I1 HLA antigens. In this report, we describe a child with acute idiopathic thrombocytopenic purpura who at the time of the thrombocytopenic episode had class II (HLA-DR) antigens on his platelets. Following recovery, the HLA-DR antigens were no longer present on the platelets. We postulated that class I1 had been induced on his megakaryocytes by a cytokine such as
interferon gamma, and that the induced expression of class I1 antigens contributed to the autoimmune disorder. To substantiate this possibility we next studied class I and II antigen expression on an erythroleukaemia cell line (HEL), which has many megakaryocytic features. Following treatment of HEL cells with interferon gamma, class I expression was increased and HLA-DR antigens were induced. These observations suggest that cytokine-mediated induced HLA-DR expression may contribute to the pathogenesis of a subset of thrombocytopenias.
Class I and class I1 MHC glycoproteins function physiologically to present antigens on the cell surface for recognition by T lymphocytes (recently reviewed by Yewdell & Bennink, 1990). Antigen presented in the context of class I MHC molecules is recognized by CD8 (suppressor-cytotoxiccells) and stimulates secretion of interleukins and other cytokines by T cells. Antigen presented in the context of class I1 MHC molecules is recognized by CD4 (helper) cells initiating the production of antibody by B cells. Almost all nucleated cells in the body express class I antigens: however, class I1 expression is much more restricted and found primarily on activated macrophages, B cells, activated T cells and dendritic cells. In addition, class I1 can be induced on many other cells by interferon gamma (Basham & Merigan. 1983: Collins et al, 1984: Wong et al, 1984: Fiers et al. 1985: Niederwieser et al, 1988). The induction of class tI expression on cells not normally carrying these antigens has been postulated to lead to inappropriate presentation of self or altered self antigens with subsequent T-helper cell recognition and autoantibody production (Bottazzo et al, 1983: Editorial, 1985). Such induced expression of class II antigens has been demon-
strated on thyroid cells and pancreatic islet cells, raising the possibilitythat altered expression of membrane proteins plays a role in the pathogenesis of autoimmune thyroiditis and diabetes mellitus (Hanafusa et al. 1983: Bottazzo et al, 1985: Pujol-Borrell et al, 1986, 1987; Grubeck-Loebenstein et al. 1988). In humans, haematopoietic progenitor cells, but not their mature progeny, express HLA-DR antigens (Fitchen et al, 1981:Fakenbergetal,1984;Amatrudaetal, 1987:Buschet al, 1987). Interferon gamma can induce HLA-DR expression on normal and leukaemic human myeloid cells and on myeloid cell lines (Basham et al, 1984: Virelizier et al, 1984: Koeffler et al, 1984; Amatruda et al, 1987). In the megakaryocyte Lineage the colony-forming unit-megakaryocyte (CFU-MK) appears to express HLA-DR whereas the burstforming unit-megakaryocyte (BFU-MK) does not (Briddell et al, 1989). Mature platelets express HLA class I antigens but not HLA class I1 antigens (Rabellinoet al, 1979; Klein, 1986: Kunicki. 1988). Loss of HLA-DR by megakaryocyte precursors may occur before the gain of the glycoprotein IIb/IJIa antigen (Koike et al. 1987; San Miguel et al. 1987). We hypothesized that transient induction of class II antigens on megakaryocytes by cytokines such as interferon gamma (perhaps stimulated by a viral infection) might underlie certain acute immune thrombocytopenias.
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Correspondence:Dr L. K. Boshkov, Room 4B2.16.Walter Mackenzie Centre, University Hospital, 8440 112 Street, Edmonton. Alberta, Canada T6G 2B7.
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HLA-DR Expression by Platelets in Acute ITP In this report we describe a child with an antecedant upper respiratory infection and a peripheral smear compatible with viral infection who presents with acute idiopathic thrombocytopenic purpura (ITP). Class 11-HLA-DK antigens were present on the child’splatelets at the time of the thrombocytopenia. The expression of these antigens was lost as the child recovered. We propose that induced expression of class I1 antigens on platelets might be involved in the pathogenesis of the immune thrombocytopenic disorder in this child. In vitro studies of the cell line HEL, an erythroleukaemia line with megakaryocytic features, demonstrated that these cells can express class I1 antigens following stimulation with interferon gamma. CASE REPORT A 4-year-old boy presented with a 1-d history of petechiae
and bruising following a respiratory tract infection approximately 10 d prior to admission. Physical examination demonstrated a diffuse petechial rash and generalized purpura without lymphadenopathy or splenomegaly. The platelet count was 5 x 109/1, white count was 4 x 10y/l with granulocytopenia (granulocytes 1 .O x 1 0 y / l ) and reactive lymphocytes. The haemoglobin was 12.7 g/dl. The direct antiglobulin test and antinuclear antibody were negative. C3, C4 and quantitative immunoglobulins were normal. The bone marrow demonstrated increased numbers of megakaryocytes. The patient was treated with 4 mg/kg of prednisone per day and the platelet count rose to normal over the next week and a half. An older sister of this child had autoimmune haemolytic anaemia and immune thrombocytopenia at age 4 months that was treated and cured by splenectomy at age 2. MATEKIALS A N D METHODS Monoclonal antibodies used in platelet and cell culture studies. Antiplatelet antibodies against GP Ilb/lIIa (Kaj-1) and GP Ib/ IX (Beb-1) were produced as described (Horsewood et al, 1991). Monoclonal antibody against the platelet Fc receptor (IV.3)(Kosenfeld et al, 1985 ) was a gift of Dr Clark Anderson, Ohio State University Columbus, Ohio. Antibodies were fluorescein isothiocyanate (FITC) conjugated to fluorescein/ protein ratios of 7.4 (anti-IIb/IIIa). 2.4 (anti-Ib/IX) and 6.1 (anti-Fc receptor). Phycoerythrin (PE)-conjugated anti-HLADR (I3),anti-CD4 (T4).and isotype IgG2a control (MS IgG2a) were obtained from Coulter Immunology, Burlington. Ontario, Canada. 13 is a nonpolymorphic class I1 (HLA-DR)Ia antigen of 34.29 kD molecular weight (Toddet ul, 1984).PEconjugated anti-HLA-DR (IOT2a)was obtained from AMAC/ Bio/Can, Mississauga, Ontario. IOT2a reacts with a monomorphic class I1 epitope (Kebai et al. 1983). Monoclonal antibody against a monomorphic determinant on HLA-A,B.C (W6/32) was obtained as a hybridoma from American Type Culture Collection (Rockville. Md.) and purified using Staph. protein A. Monoclonal antibody IgG2a isotype control (UPC 10) was obtained from Sigma, St Louis, Mo. Unconjugated murine monoclonal antibodies were visualized by the addition of FITC-conjugated F(ab)’2 adsorbed goat anti-mouse antibody (Cappel. West Chester, Pa.). Flow cytometric analysis oJ’platelets. (1) Patient and control
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platelets. Flow cytometric analysis was performed using platelets from the child collected on days 3. 8 and 21 following initiation of corticosteroid therapy, when the platelet count was 19. 42 and 1 6 7 x 109/1respectively. Platelets from a normal adult control were processed in parallel and adjusted to platelet count comparable to patient platelets with autologous platelet poor plasma before antibody binding. Additional control platelets from an adult with chronic ITP (platelet count 5 5 x 10y/l) were studied in parallel with the patient’s day 8 platelets. Other subjects who were studied included 25 normal children, six normal adults, four children with chronic ITP, seven adults with chronic ITP, and one child and one adult with acute ITP. ( 2 ) Antibody binding. Whole blood was collected into EDTA, allowed to settle at 1 g for 12 h. and the platelet rich plasma aspirated. Aspirated plasma had less than 0.8%white blood cells. Antibody binding was done directly in platelet rich plasma with 3-5 x lo6 platelets in a total reaction volume of 100 pl. The platelets were mixed separately with the FITC-conjugated monoclonal antibodies against GP IIb/ IIIa (Raj-l),GP Ib/IX (Beb-l), the platelet Fc receptor (IV.3), and with the PE-conjugated monoclonal antibodies against HLA-DR (13)and CD4 (T4).The monoclonal antibodies were used at supersaturating concentrations. Anti-HLA-DR and anti-CD4 do not normally bind to platelets and were added in quantities recommended by the manufacturer as sufficient to saturate 1 x 10‘ test white blood cells. Both a FITC-conjugated antiplatelet antibody and a PE-conjugated antibody were added simultaneously to some samples. Antibodies were allowed to bind to platelets for 1 h at 22OC. and then 1 x lo6 platelet aliquots (20-50 PI) were diluted in 1 ml of filtered calcium-albumin-free Tyrode’s buffer ( 137 mmol/l NaCI. 12 mmol/l NaHC03, 2.6 mmol/l KCI. 2 mmol/l MgCI2. 5.6 mmol/l glucose, pH 7.4) and 1 5 000 events analysed using a Coulter EPICS V flow cytometer. Platelets were identified by their forward and side light scatter profiles. Gates were set to exclude red cells and white cells. Quadrant analysis (green fluorescence on the abscissa and red fluorescence on the ordinate) was performed using Coulter software on platelet samples stained with both FITC- and PE-conjugated antibodies. H E L cell cultures and interferon gamma induction. HEL. a human erythroleukaemia cell line with megakaryocytic features (Martin & Papayannopoulou. 1982; Breton-Gorius & Vainchenker, 1986) was obtained from American Type Culture Collection (Rockville, Md.). Cells were cultured in KPMI 1640 medium with 10%heat-inactivated fetal bovine serum (Gibco Canada, Burlington. Ontario) and maintained in exponential growth. Human recombinant interferon gamma (Amersham, Oakville. Ontario, Canada) was added at the time of cell passage (initial cell concentration 1 x lo5 cells/ml) in doses ranging from 0 to 500 units/ml. After 3 d the cells were harvested, washed twice, and resuspended in calcium-albumin-free Tyrode’s buffer. Trypan blue staining confirmed > 90% viability before flow cytometry studies. Aliquots of 5 x lo5HEL cells in a total reaction volume of 200 pI were mixed separately with predetermined saturating concentrations of FITC-conjugated anti-GP Ib/D( (Beb-1).
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L. K. Boshkov, J. G. Kelton and P. F . Halloran
LOG GREEN FLUORESCENCE (FITC ANTIGCIUIX)
LOG FLUORESCENCE (FITC ANTIGCIWIX)
4 4 LOG GREEN FLUORESCENCE EFlTC ANTIGCIMXI
Fig 1. Quadrant analysis of the staining of patient day 8 platelets and platelets of a normal adult control run in parallel. Green fluorescence representing staining with the FITC-conjugated platelet-specific anti-GP Ib/M monoclonal antibody (Beb-1) is on the abscissa and red fluorescencerepresenting staining with the PE-conjugated anti-DR monoclonal antibody (13) or the PE-conjugated anti-T4 control antibody is on the ordinate. (a) Patient day 8 platelets stained with FITC anti-GP Ib/IX and PE anti-DR. (b) Patient day 8 platelets stained with FITC anti-GP Ib/rX and PE anti-T4. (c) Normal adult platelets stained with FITC anti-GP Ib/IX and PE anti-DR. A subpopulation of patient platelets showed unique positivity for the DR antigen.
anti-GP IIb/IIIa (Raj-1). and anti-platelet Fc receptor (IV.3) and with PE-conjugated anti-HLA-DR antibodies (I3 and IOTZa), anti-CD4 (T4) and isotype IgG2a control (MS IgG2a). Unconjugated anti-HLA-A,B,C (W6/32) and isotype IgG2a (UPC 10) were also mixed with HEL cells, followed by FITC F(ab)’, goat anti-mouse antibody (added 15 min after the primary antibody). Antibodies were allowed to bind for 30 min on ice. Samples were washed with 10 volumes of buffer, resuspended in 400 p1 of buffer, and 5000 events analysed per sample using a FACScan flow cytometer (Becton-Dickinson Immunocytometry Systems, Mountainview, Calif.). The results were expressed as histograms of log cell fluorescence intensity on the abscissa and cell number on the ordinate. RESULTS Platelet studies Patient day 3, 8 and 2 1 platelets stained strongly positive
with platelet-specific monoclonal antibodies against GP IIb/ m a and GP Ib/IX. A subpopulation of day 3 and 8 patient platelets were also strongly positive for HLA-DR antigen using the monoclonal antibody I3 (Fig l a , Table I). Patient platelets did not stain with PE anti-T4 (Fig lb) although this antibody stained lymphocytes appropriately. Quadrant analysis c o n b e d that the patient’s HLA-DR positive particles (platelets) were also positive for platelet-specific monoclonal antibodies (Fig l a ) excluding contamination by non-platelet particles (Ault, 1988). Normal adult platelets and platelets from an adult patient with chronic ITP tested in parallel with patient day 8 platelets showed positive staining with the monoclonal antibody against GP Ib/IX, but did not react with the monoclonal antibody against HLA-DR (Table I, Fig lc). Patient and control platelets demonstrated similar staining with the FITC-conjugated monoclonal antibody against the platelet Fc receptor (IV.3).As the child recovered,
HLA-DR Expression by Platelets in Acute ITP Table 1. Platelet positivity for anti-DR antibody staining in patient and controls.
% gated platelets Platelet count x 10'/1 mean (range)
n
positive for FITC-anti GP Ib/IX and PE-anti DR mean (range)
Patient Day 3 Day 8 Day 21
19 42 167
Adult chronic ITpt Normal adultt
55 214
1.13 0.26 0.55 (0-2.48) 0.25 (0-1.0)
39.4' 22.8 1.8
Normals Children (age 1-9) Adults
25 6
322 (250-463) 271 (214-350)
Chronic ITP Children (age 5-1 3 ) Adults
4 7
58 (1 1-109) 57 (6-1 32)
0.99 (0.04-1.70) 1.02 (0.04-2'64)
Acute ITP Children (age 3) Adults
1 1
12 10
4.16 3.88
Day 3 patient platelets were stained with FITC-anti GP IIb/IIIa and PEanti DR. t Run in parallel with patient day 8 platelets.
the percentage of platelets carrying the HLA-DR antigen progressively declined (Table I). Identically processed platelets from a further 2 5 healthy pediatric controls, six normal adult controls, four pediatric patients with chronic ITP. and seven adult patients with chronic ITP were tested. All platelets showed strongly positive staining with the anti-GP Ib/IX monoclonal antibody
555
but none was significantly positive for HLA-DR antigen. Two additional thrombocytopenic patients with acute R P were tested, one pediatric and one adult. Both were negative for HLA-DR antigen (Table I).
Induction ofclass I and I1 on HEL cells by gamma interferon HEL cells, like platelets, stained strongly positive using antiGP Ib/IX (Beb-1). anti-GP IIb/IIIa (Raj-1). and anti-platelet Fc receptor (IV.3) antibodies. HEL cells also stained strongly positively with the anti-class I antibody (W6/32). Uninduced HEL cells stained only weakly with the PE-conjugated antiHLA-DR monoclonals (I3 and IOT2a) when compared with a PE-isotype IgG2a control (MS IgG2a). Anti-CD4 (T4)was not reactive. Following treatment with interferon gamma, class I surface antigen expression increased as shown by increased W6/32 binding (Fig 2a). Induction of surface HLA-DR antigen also occurred (Fig 2b) as shown by comparable increases in binding of both anti-HLA-DR antibodies. Induction of both class I and class I1 HLA-DR showed a dose response and was maximal at an interferon concentration of 200 pg/ml. The interferon gamma stimulation experiments were performed on two separate occasions with the same results. Binding of anti-GP Ib/IX, anti-GP IIb/IIIa. and isotype controls was not changed following interferon treatment (data not shown). Cell viability as assessed by trypan blue exclusion was not affected by interferon treatment, nor did interferon treatment cause appreciable morphological changes as assessed visually or by changes in forward and side scatter parameters flow cytometrographically. DISCUSSION Idiopathic thrombocytopenic purpura (ITP) is a common autoimmune disease with two general patterns of presentation and chronicity. Most adults with ITP have chronic disease with an insidious onset, whereas young children tend to have an acute onset of self-limited thrombocytopenia which often is preceded by a viral infection. Although the
f 2QB
4Q0
CQP
OPQ
RED FLUORESCENCE Fig 2. Response of HEL cells to interferon gamma (200 pg/ml):-, untreated: ....,IFN 200. (a)Interferon gamma increases surface expression of HLA class I antigen as shown by increased binding of the anti-HLA-A.B,Cmonoclonal antibody W6/32 visualized by FITC-conjugated F(ab)'2 goat anti-mouse antibody. (b) Interferon gamma induces surface expression of HLA-DR as shown by increased binding of PE-conjugated monoclonal antibody 13.
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L. K. Boshkov, 1. G. Kelton and P. F. Halloran
target antigens of ITP have been identified as platelet glycoproteins (most often glycoprotein IIb/IIIa) (Karpatkin, 19 8 5). the underlying pathogenesis of autoantibody formation remains uncertain and may be heterogeneous. We postulated that transient induced class I1 expression by platelets might explain certain acute immune thrombocytopenias and perhaps be the initiating event in chronic antibody-mediated thrombocytopenia in patients unable to limit autoantibody formation. Using flow cytometric analysis with monoclonal antibodies, we found that class I1 DR antigens were not normally present on platelets from normal adults and children, nor were they present on the platelets from a number of adults and children with chronic ITP.However, the child described here had an acute episode of ITP in which HLA-DR antigens were detected on the surface of the platelets during the acute thrombocytopenic episode. As the child recovered, the proportion of platelets carrying antigens declined to progressively lower numbers. We were careful to determine that the HLA-DR antigen expression on the child’s platelets was not due to technical factors such as contaminating white cells. the non-specific adsorption of antibody molecules, or the binding of monoclonal antibody to platelet Fc receptors. Unfortunately we have only been able to test one additional thrornbocytopenic child with acute ITP. This second child’s platelets were negative for HLA-DR (Table I). Thus the incidence of HLA-DR positivity on platelets in acute childhood ITP is unclear, and it is possible our patient is unique. To demonstrate the feasibility of induction of HLA-DR expression on megakaryocytes. we studied HLA antigen expression on the HEL erythroleukaemia cell line which has megakaryocytic features. Analysis of HEL cells demonstrated that class I antigens were present in large numbers on the cell surface. In the uninduced state there was little class I1 expression on the HEL cells. However, following the addition of interferon gamma, class I expression was increased and class I1 HLA-DR expression was induced. Induction of HLADR on HEL cells by interferon gamma was also noted by Amatruda et a1 (1987).Very recently increased class I mRNA expression and induction of HLA class I1 mRNA expression by interferon gamma was reported on another human megakaryocytic cell line, Dami (Monte et al. 1991). HLA-DR is expressed on early megakaryocyte progenitors (CFU-MK) (Briddell et al, 1989) but is lost subsequently (Koike et al, 1987: San Miguel et al, 1987). Although the role of class I1 products in antigen presentation is well established, their role in megakaryopoiesis is unclear. Class I1 expression by mature platelets has not been previously reported. While the transient expression of HLA-DR antigens on the platelets from this child with acute ITP could have been coincidental, it is our hypothesis that in this case (and perhaps others) an acute viral infection or other immunological insult generated cytokines including interferon gamma. These cytokines cause the megakaryocytes/platelets to express class I1 antigens resulting in the inappropriate presentation of platelet-specific protein to the immune system with subsequent autoantibody production and antibodymediated destruction of platelets. Recovery from the infection results in the loss of the class I1 antigen from the surface of the
platelets with resolution of the immune thrombocytopenia. For the present, the possible role of megakaryocytes as antigen-presenting cells, the extent and in vivo significanceof induction of class I1 antigens on megakaryocytes by cytokines, and the significance of platelet HLA-DR expression remain speculative. ACKNOWLEDGMENTS We thank Barbara Kurc Bagnarol for performing the quadrant analysis of platelet samples on the Coulter EPICS V and Peter Horsewood for FITC-conjugation of the anti-platelet antibodies. These studies were supported in part by a grant from the Medical Research Council of Canada and by grant 71 76 from the Special Services and Research Development Fund of the University of Alberta to L.K.B. This work was presented in part at the American Society of Hematology Meeting in Denver, Colorado, December 1991 (Boshkov et al, 1991). REFERENCES Amatruda, T.T., Bohman. R., Ranyard. j. & Koeffler. H.P. (1987) Pattern of expression of HLA-DR and HLA-DQ antigens and mRNA in myeloid differentiation. Blood. 69, 1225-1236. Ault. K.A. (1988) Flow cytometric measurement of platelet-associated immunoglobulin.Pathology and Immunopathology Research. 7, 395-408. Basham. T.Y. & Merigan. T.C. (1983) Recombinant interferongamma increases HLA-DR synthesis and expression. journal of Immunology. 130, 1492-1494. Basham. T.. Smith. W.. Lanier, L.. Morhenn. W. & Merigan. T.T. (1 984) Regulation of expression of class 11 major histocompatibility antigens on human peripheral blood monocytes and Langerhans cells by interferon. Human Immunologu. 10, 83-93. Boshkov, L.K.. Kelton. J.G. & Halloran. P.F. (1991) HLA-DR expression by platelets in acute thrombocytopenia.Blood, 78, (10). Suppl. 1, Abstract 574. Bottazzo, G.F., Dean. B.M., McNally. J.M.. MacKay, E.H., Swift, P.G.F. & Gamble, D.R. (1 985) In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulinitis. New England journal of Medicine, 3 13, 3 5 3360. Bottazz~.G.F..Pujol-Borrell,R.. Hanafusa. T. & Feldmann. M. (1983) Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity. Lancet, ii, 1 1 1 5-1 119. Breton-Gorius. J. 81 Vainchenker, W. (1986) Expression of platelet proteins during the in witro and in vivo differentiationof megakaryocytes and morphological aspects of their maturation. Seminars in Hematology. 23, 43-67. Briddell, R.A.. Brandt, J.E., Straneva. J.E.. Srour. E.F. & H o h a n , R. (1989) Characterizationof the human burst-forming unit-megakaryocyte. Blood, 74,145-151. Busch, F.W.. Langer, M.. Pawelec. G., Ziegler. A., Wernet, P., Buhring. H.J., Meyer, P. & Muller. C. (1987)HLA-class I1 antigens on human haematopoietic progenitors. Blut. 54, 179-1 88. Collins, T.. Korman, A S . . Wake, C.T..Boss, J.M.. Kappes, D.J..Fiers. W.. Auk K.A.. Gimbron, M.A., Jr, Strominger, J.L. & Pober. J.S. (1984) Immune interferon activates multiple class I1 histocompatibility complex genes and the associated invariant chain in human endothelial cells and dermal fibroblasts. Proceedings o/ the National Acudemu of Sciences of the United States of America, 81, 491 7 4 9 2 1 .
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