Characterization of a human granulocyte differentiation antigen (CDw ...

Bioscience Reports 5, 933-941 {1985) Printed in Great Britain

933

Characterization of a human granulocyte differentiation antigen (CDw 15) commonly recognized by monoclonal antibodies

Noirin C. McCARTHY, Merete T. ALBRECHTSEN and M. A. KERR Department of Pathology, University of Dundee, NineweIls Hospital and Medieal School, Dundee, U.K. (Received 12 August 1985)

Many different anti-human granulocyte monoclonal antibodies recognize the same carbohydrate antigen which contains the trisaccharide 3-fucosyl-N-acetyllactosamine. The antigen is expressed mainly on two cell surface glycoproteins of molecular weights around 105 K and 160K which are apparently not members of the LFA-1 family of proteins. Although specific for granulocytes in blood, the antigen is expressed on a wide range of non-haemopoietic cell types.

The study of lymphocyte differentiation has been greatly facilitated by the use of monoclonal antibodies raised against cell membrane or protein fractions which have allowed the identification of functional subpopulations or states of activation of the cells and the purification of specific membrane molecules. So far, this approach has been much less successful for granulocytes. Although several specific receptors for chemotactic peptides and for opsonins have been identified and deficiencies in neutrophil function have been correlated with the lack of specific surface glycoproteins, relatively little is known about the composition of the granulocyte plasma membrane. Because of the abundance of intracellular membrane bound organelles and the lack of well characterized membrane markers only partially purified membrane fractions have been obtained. The picture is complicated even more by the apparently rapid exchange of glycoproteins from intracellular pools to the cell surface. A large number of monoclonal antibodies have been generated in many laboratories which are specific for granulocytes and can be used in the study of granulopoiesis and as markers for teukaemias (see for example, Bernard et al., 1984). These anti-granulocyte monoclonals which have been produced from mice immunized with peripheral blood granulocytes, or more frequently myelomonocytic cell lines, are almost all IgM and several have been reported to recognize the same carbohydrate antigen present on both glycolipids and glycoproteins. This antigen has been shown to involve the trisaccharide 3-fucosyl-N-acetyllactosamine (Huang et al., 1983; Gooi et al., 1983). The glycoprotein antigen comprises polypeptide chains of around 145 K and 105K molecular weight (Skubitz et ai., 1983). Several IgG monoclonal antibodies which inhibit neutrophil function have also been reported (Cotter

934

McCARTHY ET AL.

et al., 1981a, b; Vadas et al., 1985). Again the antigens recognized were around 100 K molecular weight. In addition to these granulocyte specific antigens, a family of glycoproteins present on granulocytes but also on monocytes and some lymphocytes have been identified using monoclonal antibodies. These molecules termed the LFA-1 family (Sanchez-Madrid et al., 1983) share a common beta chain of molecular weight 105 K and have different alpha chains of molecular weight 150K, 165K and t 7 7 K . All appear to be involved in leucocyte adherence functions. The molecules have b e e n termed LFA-1, Mac-1 or OKM1 and gp150,95. Mac-1 is apparently the CR3 complement receptor. Deficiency of these proteins has been associated with defective granulocyte function and recurrent infection (Springer et al., 1984). In this short review we will report some of the properties of 'granulocyte specific' monoclonal antibodies raised in this laboratory which recognize the 3-fucosyl-N-acetyllactosamine structure and can be ascribed to the cluster of antibodies recognizing the antigen CDwl5 defined by the 1st International Workshop on Human Leucocyte Differentiation Antigens (Bernard et al., 1984). We will describe the widespread distribution of the antigen in non-haemopoietic tissues and present evidence to indicate its difference from the LFA-1 family of molecules. Characterization of Monocional Antibodies MC1-4 Four monoclonal antibodies MC1-4 were produced from Balb-c mice immunized three times intraperitoneally and intradermally with purified human granulocytes. They were selected by their ability to recognize granulocytes but not peripheral blood mononuclear cells, erythrocytes or platelets. The antibodies are all IgM. Analysis of the purified antibodies by SDSpolyacrylamide gel electrophoresis showed MC1 and MC2 to contain heavy and light chains of similar mobility. MC3 and MC4 had similar mobility but different from MC1 and MC2. Radioimmunoassays suggested differences in affinity between MC1 and MC2 and MC3 and MC4. Each of the antibodies recognized only neutrophil granulocytes, either polymorphonuclear or stab cells. Eosinophils or monocytes stained for esterase activity using alpha naphthyl butyrate were not recognized. Cytofluorimetric analysis showed > 90% of neutrophils from I 0 individuals were recognized. The binding of radiolabelled monoclonal antibodies MC1 and MC4 to peripheral blood neutrophils is shown in Fig. 1. The antibodies showed different avidity but at saturation both bound 3.5 X 104 molecules per granulocyte. When bound at saturation in t h e presence of excess unlabelled antibody the rate of dissociation of cell bound antibody was slow, following first order kinetics over 16 h allowing the calculation of dissociation rate constants of 7.7 • 10-6 s for MC1 and 3.3 • 10-s s for MC4. This slow dissociation which probably reflects multivalent binding of the IgM means that loss of antibody in washing steps during immunoassays is negligible. This allows calculation of the number of molecules bound at saturation and the avidity of the antibodies. The high avidity and slow dissociation rates also means that meaningful competition studies can be made with IgG monoclonal antibodies.

GRANULOCYTE DIFFERENTIATION ANTIGENS

93 5

28

24

20 5

e" v

E"e'.,9

16

e"

4

X

12

3

i

I

I

I

i

i

1

2

3

4

5

6

Monoclonal I~M added ng. x 10 "r

Fig. 1. Direct binding of 12SI-labelled monoclonal antibodies. Duplicate samples of freshly prepared granulocytes (1 x 10 s cells) were incubated with I125-1abelled monoclonal antibody MC1 (o) or MC4 (A) in precoated vinyl plates for 2 h at 4~ The cells were then washed three times with 0.5% BSA/PBS and the radioactivity counted after drying. When the radiolabetled monoclonal antibodies were incubated at saturation in the presence of a ten-fold excess of unlabelled monoclonal antibodies MC1-4, FMC10 or 12, or VIMD5, less than 0.5 x 104 c.p.m. were bound.

The binding of MC 1 or MC4 was markedly inhibited by excess of unlabelled MC1, 2, 3 or 4 but not by the IgM fraction from ascites produced by the parent NS1 cell line or normal mouse IgM. The binding was also inhibited by anti-granulocyte monoclonal antibodies FMC10, FMC12 and VIMD5 produced in other laboratories (Zola et al., 1981; Majdic et al., 1981) which have been assigned to the cluster CDwl 5. It is therefore likely that a large number of different antibodies recognize this antigen. In an ELISA system using partially purified granulocyte membrane as target and class specific second antibodies, the binding of MC1 did not, however, inhibit the binding of monoclonal antibody OKM1 or monoclonal antibody MHM 23, which recognizes the beta chain of the LFA-1 molecule.

Characterization of the Antigen Recognized by MC1-4 The antigen recognized by these monoclonal antibodies was detectable following fixation of neutrophils by standard techniques in ethanol,

936

McCARTHY ET AL.

methanol, glutaraldehyde or formaldehyde followed b y paraffin embedding o f tissues at 60~ The antigen in partially purified membrane fractions was also stable to boiling for 30 min (Fig. 2). This marked stability is consistent with the antigen being carbohydrate. The antigen was, however, labile to treatment with 0.1N NaOH at 37~ which is usually taken to indicate an O-linked carbohydrate structure. The reactivity o f the monoclonal antibodies with purified glycoproteins and oligosaccharides was the same as that of other anti-granulocyte antibodies which have been shown to recognize 3-fucosyl-N-acetyllactosamine (T. Feizi, personal communication).

e-

e~

X

I

I

|

I

i

1/3

1/9

1/27

1/81

1/243

I

ABSORBING TISSUE DILUTION

Fig. 2. Stability of the antigen recognized by MC1-4. A partially purified membrane fraction was incubated at 4 C in PBS for 30 mm ( ) or boiled for 30 rain (a) or treated at 37~ for 48 h with 0.1 N NaOH (u). Residual antigen was measured by its ability to absorb monoclonal antibody MC2 in a trace binding assay. 9

.

o

.

.

9

The marked stability of the antigen also allowed detection b y immunoblotting after separation o f granulocyte membrane proteins b y SDS-gel electrophoresis under either reducing or non-reducing conditions (Fig. 3). The antigen is associated with t w o broad bands corresponding to polypeptides in the molecular weight ranges around 105 K and 1 6 0 K , the lower band frequently appearing as a doublet. The broad bands are probably indicative of peptides with a variable degree of glycosylation. On longer incubation o f the immunoblots with substrate minor higher molecular weight bands did appear. Occasionally, a polypeptide of molecular weight around 70 K was also observed which was believed to be a product o f protOolysis. The results confirmed that the antigen is associated with b o t h o f the polypeptide chains which have been shown to be immunoprecipitated b y


937

Fig. 3. Immunoblotting of the glycoprotein antigens recognizedby MC1-4. The proteins from the total membrane fraction of granulocyteswere separated by SDS-gel electrophoresis and then blotted onto nitrocellulose.The blot is shown stained for protein (a, b) and by immunoperoxidasefor antigenMC1(c).

similar monoclonal antibodies (Skubitz et al., 1983). Recently, the structures of two lactosaminoglycans from human granulocytes which contain 3-fucosylN-acetyllactosamine have been determined (Spooncer et al., 1984; Fukuda et al., 1984). It is assumed that these oligosaccharides carry the CDwl5 antigen. In addition to its association with two glycopeptides, the antigen is also apparently associated with glycolipids in that monoctonal antibodies MC1-4 could be absorbed completely by a chloroform-methanol extract of granulocyte membranes. This is consistent with previous reports showing the binding of similar monoclonal antibodies to granulocyte glycolipids separated by t.l.c. However, comparison of the binding of a glycolipid extract and binding of detergent solubilized protein extracts suggest that less than 10% of the antigen is lipid bound.

Localization of the antigen in lymphoid and non-lymphoid tissues by immunoperoxidase techniques The marked stability of the antigen to fixation techniques has allowed detailed study to be made of the tissue distribution of the antigen in paraffin

938

McCARTHY ET AL.

embedded sections. Monoclonal antibodies MC1-4 gave very little staining of lymph node except for a few neutrophils in blood vessels. Similarly, in spleen, staining was restricted to neutrophils in the red pulp. All neutrophils in all tissues were stained but macrophages and accessory cells of the lymphoid tissue and other tissue macrophages such as Kupffer cells and alveolar macrophages were not. In addition to neutrophils the antigen has, however, been detected on a number of other celt types. The details o f an extensive survey of the localization of the antigen in non-haemopoietic cells has been reported for these antibodies (McCarthy et al., 1985) and for two other antibodies of apparently similar specificity (Schienle et al., 1982; Howie et al., 1984). The antigen was detectable on mucins of the gastrointestinal tract, cells of the kidney and a restricted population of cells in the stomach but not on any cells in lung. The antigen was, however, expressed in large amounts on cells of these tissues in the fetus and on tumours of these tissues. It is therefore not surprising that many monoclonal antibodies raised against tumours share the same specificity (see Kerr & McCarthy, 1985). The antigen is also expressed on several cell types of neuroectodermal origin including cells of the adrenal medulla, the pituitary and, in brain, on a subpopulation of astrocytes (Fig. 4a) and on the processes of cells in the cerebellum. From chemical analysis, it is apparent that the 3-fucosyl-N-acetyllactosamine is expressed even more widely but not recognized by these granulocyte specific monoclonal antibodies. Some antibodies which apparently show the same specificity do recognize an antigen expressed on cells of the monocyte lineage. Treatment of tissues with neuraminidase appears to expose some of these hidden determinants (Howie & Brown, 1985). Interestingly, although the epitope is not revealed on monocytes, neuraminidase treatment does reveal the antigen on Langerhans cells in the skin (Fig. 4b) and possibly on dendritic cells in some lymph nodes. Subcellular localization o f the antigen on granulocytes In spite of the widespread tissue distribution of the antigen, the limited localization in blood cells to two neutrophil glycoproteins does suggest the possibility of a specific role for the glycoproteins in neutrophil function. Immunohistochemical staining of neutrophils in tissues has consistently shown only surface staining. When a preparation of neutrophils was fixed using Karnovsky's fixative (Karnovsky, 1965) and embedded in epoxy resin only surface labelling was observed on sectioning and immunoperoxidase staining (Fig. 4c), which suggested the lack of a large intracellular pool of these glycoproteins. This was confirmed by cytofluorographic studies on comparison of the labelling of untreated neutrophils and neutrophils induced to degranulate using the chemotactic peptide N-formyl Met-Leu-Phe. In contrast to the expression of the antigens recognized by OKM1 and by monoclonal antibodies recognizing the alpha and beta chains of LFA-1 which were increased markedly on stimulation, as shown by Springer et al. (1984), expression of MC1 increased only slightly. This again suggests that although the antigens recognized by the two groups of antibodies appear similar in size, the carbohydrate recognized by MC1-4 is not carried by

939

G R A N U L O C Y T E D I F F E R E N T I A T I O N ANTIGENS

c q ,..o

;~

o'~

9

o ~ .E c~ .~

~ ~ ~ ~-.~. 9 . ~

0

r~

r~ ~C3 C~

~

9

~

~-~

940

McCARTHY ET AL.

m e m b e r s o f the L F A f a m i l y o f molecules. This is f u r t h e r s u p p o r t e d b y p r e l i m i n a r y studies o n t h e g r a n u l o c y t e s o f a p a t i e n t w i t h a p p a r e n t h e t e r o z y g o u s d e f i c i e n c y o f the L F A f a m i l y o f m o l e c u l e s w h o express n o r m a l levels of MC1-4.

Acknowledgements We are m o s t g r a t e f u l to Mr. G. Coghill a n d to Mr. R. W. F a w k e s f o r assistance with t h e h i s t o l o g y and to Dr. A. J. McMichael for the gift o f m o n o c l o n a l a n t i b o d i e s . This w o r k was s u p p o r t e d b y the Medical R e s e a r c h Council.

References Bernard A, Boumsell L, Dausset J, Milstein C & Schlossman SF (1984) Leucocyte Typing, Springer-Verlag, Berlin. Cotter TG, Keeling PJ & Henson PM (1981a) A monoclonal antibody inhibiting FMLPinduced chemotaxis of human neutrophils. J. Immunol. 127,2241. Cotter TG, Spears P & Henson PM (1981b) A monoclonal antibody inhibiting human neutrophil chemotaxis and degranulation. J. Immunol. 127, 1355. Fukuda M, Spooncer E, Oates JE, Dell A & Klock JC (1984) Structure of sialylated fucosyl lactosaminoglycanisolated from human granulocytes. J. Biol. Chem. 259, 10925. Gooi H, Thorpe SJ, Hounsell EF, Rumpold H, Kraft D, Forster O & Feizi T (1983) Marker of peripheral blood granulocytes and monocytes of man recognised by two monoclonal antibodies VEP8 and VEP9 involves the trisaccharide, 34ucosyl-N-acetyllactosamine. Eur. J. Immunol. 13,306. Howie AJ & Brown G (1985) Effect of neuraminidase on the expression of 3 fucosyl N acetyllactosamine antigen in human tissues. J. Clin. Pathol. 38,409. Howie AJ, Brown G, Fisher AG & Khan M (1984) Widespread distribution in human tissues of an antigenic determinant of granulocytes. J. Clin. Pathol. 37, 555. Huang LC, Civin CI, Magnani JL, Shaper JH & Ginsburg V (1983) My-1 human myeloid specific antigen detected by mouse monoclonal antibodies is a sugar sequence found in lacto-N-fucopentaose I 11. Blood 61, 1020. Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Biol. 27, 137A. Kerr MA & McCarthy NC (1985) A carbohydrate differentiation antigen of granulocytes, brain and many turnouts. Biochem. Soc. Trans. 13,424. Majdic O, Liszka K, Lutz D & Knapp W (1981) Myeloid differentiation antigen defined by a monoclonal antibody. Blood 58, 1127. McCarthy NC, Simpson JRM, Coghill G & Kerr MA (1985) Expression in normal adult, fetal and neoplastic tissues of a carbohydrate differentiation antigen recognised by antigranulocyte mouse monoclonal antibodies. J. Clin. Pathol. 38, 521. Sanchez-Madrid F, Nagy J, Robbins E, Simon P & Springer TA (1983) A human leukocyte differentiation antigen family with distinct alpha subunits and a common beta subunit; the lymphocyte function associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1) and the p150,95 molecule. J. Exp. Med. 158, 1785. Schienle HW, Stein M & Muller-Ruchholtz W (1982)Neutrophil granulocytic cell antigen defined by a monoclonal antibody - its distribution within normal haemic and non haemic tissue. J. Clin. Pathol. 35,959. Skubitz KM, Pessano S, Bottero L, Ferrerro D, Rovera G & August JT (1983) Human granulocyte surface molecules identified by murine monoclonal antibodies. J. Immunol. 131, 1882. Spooncer E, Fukuda M, Klock JC, Oates JE & Dell A (1984) Isolation and characterisation of polyfucosylated lactosaminoglycan from human granulocytes. J. Biol. Chem. 259, 4792.


941

Springer TA, Thompson WS, Miller LJ, Schmalstieg FC & Anderson DC (1984) Inherited deficiency of the Mac-l, LFA-1, p 150,95 glycoprotein family and its molecular basis. J. Exp. Med. 160, 1901. Vadas MA, Lopez AF & Williamson DJ (1985) Selective enhancement of the expression of granulocyte.functional antigens 1 and 2 on human neutrophils. Proc. Natl. Acad. Sci. U.S.A. 82, 2503. Zola H, McNamara P, Thomas M, Smart IJ & Bradley J (1981) The~preparation and properties of monoclonal antibodies against human granulocyte membrane antigens. Brit. J. Haematol. 48,481.