3ae8: monoclonal antibody defining inflammatory macrophages in three species

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HYBRIDOMA Volume 3, Number 2, 1984 Mary Ann Liebert, Inc., Pubiishers 3AE8: Monoclonal Antibody Defining Inflammatory Macrophages in Three Species ZHANG CHEN, SHING-ERH YEN, and WILLIAM S. WALKER ABSTRACT A mouse monoclonal antibody (MAb 3AE8) of the IgGl isotype was prepared against rabbit splenocytes and was found by indirect ¡mmunofluorescence and direct binding assays to react, in the rabbit, primarily with oil-induced peritoneal exúdate macrophages (PEM0). This MAb did not bind to rabbit T cells, B cells, polymorphonuclear leukocytes, or resident alveolar or peritoneal M0, but it did bind to a subpopulation of rabbit splenocytes with surface characteristics of null cells. The antibody also recognized mouse and rat PEM0 as well as the murine M0 cell lines P388D1 and IC-21. Consistent with findings in the rabbit, it did not bind to M0 obtained from the peritoneal cavities of normal rats or mice. The addition of MAb 3AE8 to mouse PEM0 caused a marked enhancement in the phag- ocytic uptake of erythrocyte target cells sensitized with a mouse antierythrocyte antiserum. INTRODUCTION During the course of preparing mouse hybridomas secreting monoclonal antibodies (MAbs) reactive with rabbit lymphocytes, '" we noted that one hybridoma (3AE8) produced a MAb that bound primarily to rabbit peritoneal exúdate macrophages (PEM0s). Since MAbs to M0 have proven of value in defining distinct differentiation and maturation stages of mononuclear phagocyte develoment,'2"7' we studied in greater detail the cellular binding patterns of MAb 3AE8 and its effect on the antibody-dependent phagocytic funciton of M0. We report here that MAb 3AE8 appears to differ from other anti-M0 MAb described to date since it recognized an epitope common to rat, mouse, and rabbit PEM0. Furthermore, PEM0S exposed to MAb 3AE8 were markedly enhanced in their antibody-dependent phagocytic activity. MAb 3AE8 also reacted with a subpopulation of rabbit splenic leukocytes enriched in null cells. MATERIALS AND METHODS Animals Rabbits of either sex and weighing between 1.5 and 2 kg were obtained from a closed colony maintained at this institution. Female BALB/c, C3Hf/An, and C57BL/6 mice were purchased from Cumberland View Farms, Clinton, TN. Male Wistar/Furth (W/Fu) rats were obtained from Harlan Industries (Indianapolis, IN) and weighed between 180 and 200 g at the time of their use. Cell lines Hybridoma ACM-1, which secretes an anti-rabbit T-cell and polymorphonuclear leukocyte MAb of the IgGl isotype, was prepared as described elsewhere.'" Hybridoma 9AE10, which Division of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38101. 141

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Page 1: 3AE8: Monoclonal Antibody Defining Inflammatory Macrophages in Three Species

HYBRIDOMAVolume 3, Number 2, 1984Mary Ann Liebert, Inc., Pubiishers

3AE8: Monoclonal Antibody DefiningInflammatory Macrophages in Three SpeciesZHANG CHEN, SHING-ERH YEN, and WILLIAM S. WALKER

ABSTRACTA mouse monoclonal antibody (MAb 3AE8) of the IgGl isotype was prepared againstrabbit splenocytes and was found by indirect ¡mmunofluorescence and direct binding assaysto react, in the rabbit, primarily with oil-induced peritoneal exúdate macrophages (PEM0).This MAb did not bind to rabbit T cells, B cells, polymorphonuclear leukocytes, or residentalveolar or peritoneal M0, but it did bind to a subpopulation of rabbit splenocytes withsurface characteristics of null cells. The antibody also recognized mouse and rat PEM0as well as the murine M0 cell lines P388D1 and IC-21. Consistent with findings in therabbit, it did not bind to M0 obtained from the peritoneal cavities of normal rats or mice.The addition of MAb 3AE8 to mouse PEM0 caused a marked enhancement in the phag-ocytic uptake of erythrocyte target cells sensitized with a mouse antierythrocyte antiserum.

INTRODUCTION

During the course of preparing mouse hybridomas secreting monoclonal antibodies (MAbs)reactive with rabbit lymphocytes, '" we noted that one hybridoma (3AE8) produced a MAb

that bound primarily to rabbit peritoneal exúdate macrophages (PEM0s). Since MAbs to M0have proven of value in defining distinct differentiation and maturation stages of mononuclearphagocyte develoment,'2"7' we studied in greater detail the cellular binding patterns of MAb 3AE8and its effect on the antibody-dependent phagocytic funciton of M0.

We report here that MAb 3AE8 appears to differ from other anti-M0 MAb described to datesince it recognized an epitope common to rat, mouse, and rabbit PEM0. Furthermore, PEM0Sexposed to MAb 3AE8 were markedly enhanced in their antibody-dependent phagocytic activity.MAb 3AE8 also reacted with a subpopulation of rabbit splenic leukocytes enriched in null cells.

MATERIALS AND METHODS

AnimalsRabbits of either sex and weighing between 1.5 and 2 kg were obtained from a closed colony

maintained at this institution. Female BALB/c, C3Hf/An, and C57BL/6 mice were purchasedfrom Cumberland View Farms, Clinton, TN. Male Wistar/Furth (W/Fu) rats were obtained fromHarlan Industries (Indianapolis, IN) and weighed between 180 and 200 g at the time of their use.

Cell linesHybridoma ACM-1, which secretes an anti-rabbit T-cell and polymorphonuclear leukocyte

MAb of the IgGl isotype, was prepared as described elsewhere.'" Hybridoma 9AE10, which

Division of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38101.

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CHEN ET AL.

secretes an IgM MAb with specificity similar to ACM-1, was provided by Dr. K. Knight.'8' Themouse macrophage-like cell lines P388D1 and IC-21 have been fully characterized, and proceduresfor their growth and harvesting are described elsewhere.'9, ""

Preparation of hybridoma 3AE8A BALB/c mouse was immunized by i.p. injection of a suspension of 2 x 10' rabbit spleen

cells enriched for B cells by prior treatment with MAb 9AE10 and complement (see below). Theanimals received boosters three weeks later with a similar injection and a suspension of themouse spleen cells was prepared four days later.

The murine splenic leukocytes were fused with SP2/0 myeloma cells using 35% (w/v) poly-ethylene glycol, essentially as described by Gefter and coworkers."" Hybrids were selected inHAT medium containing BALB/c resident peritoneal cells as feeder layers. Supernatant fluidfrom wells containing hybridomas were screened for antibody activity by indirect immunoflu-orescentmicroscopy (see below), and those of interestwere cloned by the limiting dilutionmethod"2'in cultures containing normal rat thymocytes as feeder cells.

The MAbs in tissue culture fluids were typed by immunodiffusion using H-chain specific reagents(Litton Bionetics, Kensington, MD). Ascitic fluid was harvested from Pristane-primed BALB/cmice bearing hybridoma 3AE8.

Cell suspensionsSingle cell suspensions of rabbit spleen, mesenteric lymph node, appendix, thymus, and bone

marrow were prepared as previously described.'". Rabbit PE cells, containing about 95% M0as defined by morphologic criteria, were obtained four days after animals had been injected withmineral oil (Klearol, Ruger Chemical Co., Irvington, NJ, see Ref. 13). Resident alveolar andperitoneal cells were obtained from normal rabbits by lavage. Rabbit polymorphonuclear leu-kocytes were obtained from animals 4 h after i.p. injection of 50 ml of 0.1% glycogen in saline."4'

Mouse PE cells were obtained four days after i.p. injection of 1 ml of Brewer's thioglycolatebroth and resident peritoneal cells were lavaged from normal mice."5' Bone marrow cells were

obtained from femurs as previously described."5' All suspensions of cells were washed and re-

suspended in medium RPMI 1640 containing 10% fetal bovine serum (FBS).

Culture-derived bone marrow macrophagesMouse femurs were removed intact and the bone marrow cells (BMC) were flushed from the

cavity with 1 ml of alpha-MEM (Grand Island Biological Co., Grand Island, NY). The cells were

dispersed and resuspended in alpha-MEM containing 10% FBS and 10% L-cell-conditioned me-

dium, which served as a source of colony-stimulating factor l."6' Suspensions containing 3 x

10" BMC in 3 ml of complete alpha-MEM were plated into 35 mm dishes containing glass coverslipsand the cultures were incubated at 37°C in a humidified atmosphere of 5% C02 in air.

Immunefluorescence assayThe indirect immunofluorescent microscopic assay was performed as previously described'"

except that the FITC-conjugated F(ab)2 fragment of a goat anti-mouse F(ab)2 antibody was usedas the second reagent. The conditions of the assay were optimal with regard to dilutions of bothantibody reagents, as determined in preliminary experiments.

Separation of T, B, and null cellsSuspensions of rabbit spleen cells were enriched for T cells by the panning method of Mage

and coworkers."7' Briefly, 25 x 100 mm Petri dishes (Falcon, Oxnard, CA) were coated overnight

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MAb DEFINING INFLAMMATORY MACROPHAGES

with antibody to the Fab fragment of rabbit IgG. Unbound antibody was removed and the plateswere rinsed five times with phosphate buffered saline (PBS), pH 7.4. A suspension of nucleatedrabbit splenocytes (5 x 107 in 5 ml of RPMI 1640 containing 15% FBS) was added to each plateand the cultures were incubated stationary, except for swirling about halfway through the in-cubation period, for 1 h at 4°C. The nonadherent cells, recovered by swirling the plates, werecollected and washed in fresh medium.

Populations of splenocytes enriched for B cells were obtained by incubating cell suspensions(5 x 106/ml) with a previously determined optimal dilution of MAb 9AE10 for 30 min at roomtemperature and, after centrifugation (400 x g, 10 min), resuspending the cells in an equal volumeof undiluted fresh rabbit serum, which served as a source of complement. After 90 min at 37°C,the cells were collected by centrifugation, washed, and counted.Null cells were obtained by removing B cells by the panning method from cell suspensions

recovered after the MAb 9AE10 and complement step. A flow diagram of the procedure is pre-sented in Fig. 1.Purity of the isolated subpopulations was assessed by rosetting for surface immunoglobulin-

bearing B cells with sheep red blood cells coated with goat anti-rabbit F(ab),,"8' and by indirectimmunofluorescence for T cells using MAb ACM-1 and FITC-labeled goat anti-mouse Ig anti-serum.'"

Trypsin treatment

Cells were washed in PBS and resuspended to 1 x 106/ml in 0.1% trypsin (TPCK, Flow General)prepared in medium RPMI 1640 lacking serum. The mixtures were incubated for 15 min at 37°C,after which 1.0 ml of FBS was added and the cells were collected by centrifugation.

Preparation of the Fab fragment from MAb 3AE8Freon-clarified ascitic fluid'19' containing MAb 3AE8 was treated at 50% saturation of ammonium

sulfate to precipitate the immunoglobulins which, following dialysis, were further purified by

NUCLEATED RABBITSPLEEN CELLS

TREAT WITH ANTI-T-CELLMoAb AND RABBIT C

PAN FOR SURFACE Ig* CELLS

SUBPOPULATION ENRICHEDFOR B- AND NULL-CELLS

PAN FOR SURFACE Ig* CELLS

SUBPOPULATION ENRICHEDFOR NULL CELLS

SUBPOPULATION ENRICHEDFOR T- AND NULL-CELLS

FIG. 1. Scheme of the separation steps employed to obtain subpopulations of rabbit splenicleukocytes enriched for T, B, and null cells.

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CHEN ET AL.

DEAE chromatography. Initial attempts to prepare the F(ab)2 fragment from purified MAb 3AE8were unsuccessful with standard papain digestion procedures.'20' The Fab fragment was readilyobtained, however, when (i) the working stock solution of papain was prepared fresh and itsconcentration was increased from 1% to 2% of the IgG content, (ii) the 0.1 M 2-mercaptoethanolsolution was freshly prepared, and (iii) the incubation time was increased to 10 h. The resultantpapain digests were dialyzed against 0.1 M Tris-HCl buffer, pH 7.0, and the Fab fragments were

eluted from DEAE-AFFi-gel columns (Bio/Rad Laboratories, Richmond, CA) with 50 mM NaCIin 0.1 M Tris-HCl buffer. Completeness of the digestions and purity of the Fab fragments were

assessed by polyacrylamide gel electrophoresis.

Radiolabeling of the Fab fragment ofMAb 3AE8 and the binding assayThe Fab fragment (50 u.g) of MAb 3AE8 was labeled with 100 u,Ci ,2T (Amersham, Chicago,

IL) by the Iodogen method,'2" and free l2T was separated from labeled protein by gel filtrationon PD-10 columns (Pharmacia, Uppsala, Sweden). The dialyzed, labeled proteins had specificactivities of about 3 x 106 cpm/u.g protein, of which >90% was insoluble in 10% trichloroaceticacid.

On the day of use, aliquots of the labeled antibody were centrifuged at 105,000 x g for 15min (Beckman Airfuge) and the samples were diluted in gelatin-veronal buffer (GVB, Ref. 22).For the binding assay, 1 x 105 cells were collected in microcentrifugation tubes and resuspended

in labeled antibody, and the mixtures were incubated in an ice bath for 60 min. The cells were

then washed four times in GVB, transferred to new tubes, and the cell-associated radioactivitywas determined.

RESULTS

Tissue reactivity ofMAb 3AE8

For cells isolated from the rabbit, MAb 3AE8 reacted primarily with oil-induced PE cells and,to a lesser extent, with cells isolated from bone marrow and spleen (Table 1). This pattern ofreactivity, coupled with the failure of the antibody to bind to cells isolated from tissues containinglarge numbers of mature lymphoid elements, such as the lymph nodes and the appendix (Table1 ), suggested that MAb 3AE8 recognized an epitope(s) found on some members of the mononuclear

Table 1. Rabbit Tissue Reactivity of MAb3AE8.

%Tissue 3AE8* cells

60-7020-3020-30<5<5<1<1

Cells from the indicated tissues were tested by an indirectimmunofluorescence assay for reactivity with MAb 3AE8. Thevalues are from assays of tissues from at least six differentrabbits.

Peritoneal exúdate cellsBone marrow cellsSpleenLymph nodeAppendixThymusBlood polymorphonuclear cellsBlood erythrocytes

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phagocyte lineage(s). This notion was confirmed when a variety ofM0 populations from rabbit,mouse, and rat were examined for MAb 3AE8 reactivity (Table 2). Of particular interest wasthe finding that, regardless of the species tested, essentially all reactivity was limited to M0 inelicited inflammatory exudates. With murine M0 lines, essentially all the cells bound MAb 3AE8.

MAb 3AE8 does not bind to M0 via the Fc-fragmentImmunodiffusion analysis of culture fluids containing MAb 3AE8 revealed that the antibody

was isotype IgGl (data not shown). Murine antibodies of this isotype, particularly when aggre-gated,'231 can bind through the Fc-domain to M0 Fc-receptors. It was, therefore, important todetermine whether the binding of MAb 3AE8 to inflammatory M0s, which are enriched forFc-receptor activity when compared wtih resident M0,'24' was mediated by the Fab or Fc portionof the IgGl molecule.

Several lines of circumstantial evidence suggested that 3AE8 binding was not Fc-mediated.First, MAb ACM-1, an IgGl which reacts with rabbit T cell and PMN'" did not bind to PEM0.Second, disaggregation of solutions of MAb 3AE8 (105,000 x g, 30 min) failed to remove reactivityto PEM0. Third, the addition to the MAb 3AE8 incubation mixtures of heat-aggregated IgGisolated from normal rabbit or mouse serum, or the addition of soluble immune complexes formedbetween rabbit or mouse anti-human IgG antisera and human IgG, failed to inhibit the bindingof the MAb to PEM0.Direct evidence ruling out involvement of the Fc-fragment was obtained when the distribution

of reactivity of the purified Fab fragment from MAb 3AE8 was examined. While the intensityof fluorescence was reduced when compared with equivalent protein concentrations of intactMAb 3AE8, the patterns of reactivity were the same as those shown in Tables 1 and 2.

Binding characteristics of3AE8 to mouse and rabbit inflammatory M0The data in Table 2 show that populations of murine PEM0 contained a higher proportion of

3AE8-binding cells (>90%) than populations of oil-induced rabbit PEM0. Furthermore, it was

consistently noted that the intensity of 3AE8 staining was greater on murine M0 than on thosefrom rabbits.

To determine whether the amount of MAb 3AE8-binding epitope differed between mouse andrabbit PEM0, we added increasing amounts of '"I-labeled Fab 3AE8 to constant numbers ofcells from each species and, after incubation at 4°C for 1 h, determined the amount of cell-associated radioactivity. Figure 2 presents the results of a representative experiment and showsthat mouse PEM0 bound about three times the amount of MAb 3AE8 than did rabbit PEM0.Since the proportion of 3AE8-reactive cells in the two populations does not differ by a factor of

Table 2. Reactivity of MAb 3AE8 with Macrophages.

% 3AE8 + Macrophages fromMacrophages from Rabbit Mouse RatPeritoneal resident <5 <10 <10Peritoneal exúdate 60-70 >90 >90Lung <5 <5 <5Cell lines:

P388D1 >90IC-21 >90

Macrophages from the indicated species and tissues were cultured for 2-4h on glass coverslips and the adherent cells were assayed for their ability tobind MAb 3AE8. The values presented summarize a series of at least fiveexperiments for each tissue and species.

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20 40 60 80'

160,25I-3AE8(Fab) Added LaL)

FIG. 2. The binding of '"Mabeled MoAb 3AE8 Fab-fragment to rabbit (O-O) and mouse (•-•) peritoneal exúdate M0. The mean values from triplicate samples of a representative experimentare shown.

3 (Table 2), we conclude that mouse PEM0 contain more of the 3AE8 epitope, on a per-cellbasis, than do rabbit cells.

The expression ofMAb 3AE8-binding activity on culture-derived mouse bonemarrow M0

Populations of differentiated mouse M0 are readily obtained when BMCs are cultured in thepresence of colony-stimulating factor l."6' Since MAb 3AE8 reacts with both rabbit and mouse

PEM0 (Table 2), we determined whether fresh mouse BMC and BM-derived M0 expressedMAb 3AE8-binding activity (Table 3). In contrast to the findings with fresh rabbit BMC (Table1), only about 10% of mouse BMC reacted with MAb 3AE8. Essentially all the M0 derived fromthe BMC, however, reacted with the antibody.

Table 3. Reactivity of FreshBone Marrow Cells and

Culture-Derived MurineBone Marrow M0 with

MAb 3AE8.

% (range) MAb 3AE8* cells7-day

BMC Adherent M07(3-11) 85(80-87)

Suspensions of fresh mouse bone marrowcells (BMC) and the adherent subpopulationof 7-day culture-derived mouse bone marrow

M0 were tested for their content of MAb3AE8+ cells by an indirect immunofluoresc-ence assay. The means and ranges from threerepresentative experiments are presented.

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Table 4. Effect of Trypsin on the Expression of the3AE8 Epitope on Peritoneal ExúdateMacrophages.

% 3AE8+ M0Treatment Rabbit Mouse

None 70 85Trypsin <1 <1

Trypsin and culture 70 >90The indicated populations of peritoneal exúdate M0 were as-

sayed for 3AE8-bearing cells before and after trypsin treatment(0.1%, 15 min, 37°C) and the latter cells again after 18 h of cul-turing.

Nature of the 3AE8-binding epitope on PEM0Rabbit and mouse PEM0 were incubated for 15 min at 37°C in a 0.1% solution of trypsin and,

after resuspension in FBS and washing, the cells were examined for reactivity with MAb 3AE8.In addition, aliquots of enzyme-treated cells were resuspended in medium RPMI 1640 containing10% FBS and cultured for 18 h before being assayed with the antibody. Table 4 presents theresults from a representative experiment showing that the 3AE8 epitope was removed by trypsinand that it was re-expressed on M0 cultured overnight.Next we attempted to identify the molecule(s) on rabbit and mouse PEM0 recognized by MAb

3AE8. We surface-labeled [125I, lactoperoxidase method'25'] and metabolically-labeled (35S-methionine) the cells, but failed to detect, by autoradiographic analysis of polyacrylamide gels,any MAb 3AE8-bound material precipitated with either anti-mouse-Ig antisera or Staph proteinA. Additional attempts employing immunoblot procedures and I25I- labeled MAb 3AE8 or an

enhancing ,25I- rabbit anti-mouse IgG sandwich technique also proved futile.

Effect ofMAb 3AE8 on the antibody-dependent phagocytic activity ofmousePE and PR M0

During studies of whether MAb 3AE8 bound to M0 Fc-receptors, we added fluids from culturesof hybridoma 3AE8 to adherent PE and PR mouse M0 and, after various periods of time, tested

Table 5. MAb 3AE8 Enhances the Phagocytic Activity of Mouse PEM0.Pre-incubation Phagocytic activity (% and range) ofM0with MAb 3AE8 +/- -

time (min) MAb 3AE8 PE M0 PR M00 34(31-43) 12(11-17)

30-

ND ND+ 52(45-49) 13(11-15)

60-

44(38-48) 12(11-14)+ 66(63-70) 15(14-17)

120 40(34-44) 14(12-15)+ 72(70-74) 15(14-16)

Monolayers of M0 were treated with tissue culture fluid containing MAb 3AE8 for the indicated periodsof time. The cells were washed once and a 0.1% suspension of antibody-coated erythrocytes, prepared at a1:800 dilution of a hyperimmune antierythrocyte antiserum (HA titer = 200^00) was then added. After 60min of incubation, the proportion of cells containing at least one target cell was determined microscopically.The values are the means and ranges from a single representative experiment performed in triplicate.

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Table 6. Distribution of MAb 3AE8+ Cells in T-, B-, and Null-Cell-Enriched Subpopulations of Rabbit Spleen Cells.

„ , , . % of cellsSubpopulationenriched for T cells B cells 3AE8* cells

T-cells 74 2 34B-cells 10 62 47

Null cells 12 3 79

Suspensions of the indicated subpopulations, obtained as outlined in Fig. 1, were testedfor their content of 3AE8+ cells, T cells (MAb ACM-1), and B cells (E-anti-Rb Ig) as

described in Materials and Methods. The results are from a single representative ex-

periment. The T-cell enriched subpopulation represented 6%, the B-cell subpopulation48% and the null-cell subpopulation 2% of the cells in the original spleen cell suspension.

the M0 for Fc-receptor-mediated phagocytic activity using sheep erythrocytes (E) sensitizedwith a hyperimmune mouse anti-E antiserum (A) as indicator cells. These complexes [EA(IgG)]were prepared and tested at various dilutions of A (hemagglutination titer = 200-400). In no

case did we observe the inhibition of EA(IgG) uptake by M0 treated with MAb 3AE8, but a

marked enhancement in phagocytic activity of PEM0 was always observed when (i) E were

sensitized with subagglutinating dilutions of A and (ii) the PEM0 had been exposed to the antibodyfor at least 30 min prior to the addition of the target cells (Table 5).

These results confirm that MAb 3AE8 does not bind via its Fc-fragment to the Fc receptoron M0 and, furthermore, that the 3AE8 epitope is limited to elicited M0 since the antibody hadno effect on the phagocytic activity ofPRM0 (Table 5). Whether the phagocytic response againstsuboptimally sesitized target cells is the result of the upregulation of Fc-receptors or of an increasein general phagocytic activity of the cells is unknown at present.

MAb 3AE8 reacts with a subpopulation of rabbit splenic leukocytes enrichedfor null cells

M0s represent about 15% of the splenic leukocyte population.'26' The results presented inTable 1 show that MAb 3AE8 reacted with about 25% of splenic leukocytes, indicating that theepitope recognized by the antibody was present on cells other than to M0. To identify thispopulation of cells, we separated suspensions of rabbit splenic leukocytes into subpopulationsenriched for T cells, B cells, and cells lacking surface characteristics of either (null cells), anddetermined the proportion of MAb 3AE8+ cells in each subpopulation.

The results of a representative experiment are presented in Table 6. Note that almost 80% ofthe cells in the subpopulation depleted of T cells and B cells, and thus, presumably enriched fornull cells, reacted with MAb 3AE8. By contrast, the T- and B-cell-enriched subpopulation patternsof 3AE8 reactivity could be attributed to the presence of contaminating null cells.

DISCUSSION

MAbs that react with all known members of a cell lineage are useful for identifying and quan-titating cells in suspensions or tissues.'27"28' If, however, the reactivity of the MAb is limited toa subpopulation of cells, then it can provide a means for identifying distinct developmental stagesin a lineage or for testing whether multiple pathways exist in the development ofmorphologicallysimilar cells.MAb 3AE8 falls into this latter category since its reactivity among M0 is limited to those

present in inflammatory exudates or derived from cultures of mouse BMC. Other MAbs that

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react with inflammatory M0 have been described,15' but MAb 3AE8 appears to be unique in thatit recognizes a determinant present on PE M0 from rabbits, rats, and mice. The determinantrecognized by MAb 3AE8 also differs from Mac-2 and Mac-3 where the former is not detectableon splenic leukocytes'2' and the latter is present on PR M0.'3)

We were unable to identify the determinant on elicited M0 that binds MAb 3AE8. This difficultyhas been noted by others and is usually attributed either to the low avidity of the MAb or to theassociation of the epitope with a glycolipid.'6' Binding of MAb 3AE8 was prevented by treatingM0 with trypsin (Table 3) showing that the epitope was on the external aspect of the membrane.Although we cannot rule out the possibility that the effect of trypsin was secondary, we believe,that our failure to detect 3AE8-associated or binding molecule is the result of weak avidity ofthe antibody, a well-documented situation associated with MAbs.'6'

In addition to binding inflammatory and bone marrow-derived M0, MAb 3AE8 reacted witha subpopulation of rabbit splenic leukocytes. Using separation procedures that yield subpopulationsof rabbit lymphocytes highly enriched for T, B, and null cells, we were able to determine thatMAb 3AE8 is reactive with null cells. It is interesting and perhaps significant that other anti-M0 MAbs have also been found to react with some cells outside the presumed mononuclearphagoctye lineage.'2-5'MAbs that discriminate between bone-marrow-derived and inflammatory M0 on the one hand,

and steady-state resident M0 on the other hand, may prove to be of value in resolving importantquestions on the developmental biology of these cells. Most agree that M0s that accumulate atthe sites of inflammation are derived from circulating monocytes originating in the bone marrow

(for reviews see Refs. 24 and 29). Controversy exists, however, over the origins of resident M0.One view holds that all M0s, regardless of tissue location, originated from a common progenitorthat resides in the bone marrow.001 The opposing view holds that resident tissue M0s are au-

tonomous populations and are capable of self-renewal without drawing on the pool of circulatingmonocytes.13" The availability of a MAb that differentiates between these populations may proveof use in resolving this and other questions concerning the immunobiology of M0.

ACKNOWLEDGMENTS

This work was supported by Grant AI17979 (WSW) Grant CA23709 (F.L. Adler), Cancer CenterSupport (CORE) Grant CA21765 from the National Institutes of Health, and by ALSAC. Dr.Chen was a Special Overseas Research Fellow at St. Jude Children's Research Hospital and herpresent address is the Institute of Hematology, #2 Nanjing Road, Tianjing, People's Republicof China.

REFERENCES

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Address reprints to:Dr. William S. Walker

Division of ImmunologySt. Jude Children's Research Hospital

332 North Lauderdale,P.O. Box 318

Memphis, TN 38101

Received for publication January 24, 1984.Accepted after revisions March 30, 1984.

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