EXPERIMENTAL AND MOLECULAR PATIiOIBCY 24, 220-235 (1976)

Differentiation of Small Numbers of Mouse Peritoneal Cells into Antibody-Producing Plasma Cells in Diffusion Chambers

EMMA SHELTON, W. I. BENNETT, AND JAN M. ORENSTEIN

~boruto~ of Biocl~e~i~t~~, National Cancer ~~t~t~te, Bethesda, Ma~l~nd 20014

Received July 17, 1975, and in revised form September 17, 1975

Comparatively small numbers, 2.5-10 X lo’, of primed mouse peritoneal cells cultured in diffusion chambers, can with antigen, be induced to differentiate into plasma cells producing antibody specific to two different priming antigens, horse- radish peroxidase and sheep erythrocytes. This number of immunocytes is small enough to permit morphologic analysis of the events in differentiation. The overt re- sponse to SRBC was characterized by the appearance between 48 and 72 hr of pIasmablasts, many mitotic figures and alterations in the membrane mo~hology of many lymphocytes. Interactions of plasmablas~ and mature plasma cells with macro- phages were very prominent and after months of culture plasma cells still clustered specifically above macrophages or mesothelial cells. The response of peritoneal cells to horseradish peroxidase was more subtle than the response to sheep erythrocytes in that differentiation into plasma cells proceeded without major morphologic transi- tions. The intracellular localization of anti-HRP antibody in the perinuclear space and in patches near or on the plasma membrane of the mature plasma cell suggested discontinuous synthesis of specific antibody. In many mature plasma cells, no anti- HRP antibody could be detected.

INTRODUCTION

The development of lymphocytes into antibody-producing plasma cells is a complex, antigen-dependent event that may require the participation of three types of cells, thymus-derived T lymphocytes, bone-marrow derived B lympho- cytes and macrophages (Cooper et al. 1972). The exact nature of the coopera- tion between these cells is unknown (Nossal and Ada, 1971; Nossal, 1971; Miller, 1972). If a culture system could be devised that would permit smal1 numbers of immunocompetent cells to be followed morphologically as well as functionaIly after s~ulation with antigen, it would provide a way of monitoring not only the total “architecture” of the culture (cell loss, cell division) but it would also permit the observation of interactions between individual cells and their diffferentiation leading to the production of specific antibody. The cells most useful in such a system are the peritoneal cells of rodents that consist chiefly of lymphocytes and macrophages (Shelton et al. 1970; Rosenstreich et al. 1971). They respond with vigor to antigenic challenge (Rosenstreich et aE. 1971) and can be obtained as a steriIe suspension of free ceils by lavage of the peritoneal cavity. They represent a much less compfex cell mixture than spleen or bone marrow suspensions since the latter contain many myelopoietic and erythropoietic stem cells that also proliferate and differentiate in culture (Boyum and Borg- Strom, 1970).

220

Copyright Q

1976 by Academic Press, All rights o reproduction in any form

CELL DIFFERENTIATION IN DIFFUSION CHAMBERS 221

The usefulness of the diffusion chamber technique (Prehn et al. 1954) as an in V~VO culture method for the study of antibody production in the primary and secondary immune response is well documented (Holub and Riha, 1960; Capalbo et al. 1964; Weiler, 1965; Borella, 1971). Recent improvements in design, steril- ization and detoxification of chamber materials (Nettesheim and Makinodan, 1965; Makinodan et al. 1965; Nettesheim et al. 1966; Makinodan et al. 1967; Vann and Makinodan, 1969; Vann, 1969) made it possible at the peak of the primary response of mouse spleen cells to sheep erythrocytes (SRBC), to harvest lo4 plaque-forming cells per lo6 nucleated cells recovered from chambers. Sec- ondary responses of similar magnitude occurred when mouse spleen cells were presented with dinitrophenylated keyhole limpet hemocyanin (Segre and Segrd, 1972). In these studies, the initial inocula into chambers were in the range of lo6 to lo7 cells and recovery of the cells from the chambers was possible only after enzymatic digestion of a fibrous clot that developed after a period of cell growth. In addition, classification of the types of recovered cells was based upon identification in smears and imprints, while kinetic studies relied on recording the number of nucleated cells recovered. It was not possible to follow in situ the fate of the cells introduced into this type of chamber.

The present paper describes the culture in diffusion chambers of 2.5-10 x lo4 mouse peritoneal cells and the tracing in situ of their subsequent differentiation into plasma cells producing specific antibody after secondary challenge with two antigens, horseradish peroxidase (HRP) and sheep erythrocytes (SRBC).

MATERIALS AND METHODS

Chambers: Construction. Both double and single diffusion chambers were used in these experiments. Double diffusion chambers were made by cementing Type HA or Type TH Millipore filters (Millipore Filter Corp. Bedford, Mass.) between acrylic plastic (Lucite) rings, 1 mm thick, 2 cm outside diameter, 1 cm inside diameter (for details of chamber construction, Shelton and Rice, 1958). Two compartments were formed by gluing Schleicher and Schuell (S&S) grade B-12 Selectron nitrocellulose filters to the outside of the rings (Filters, Schleicher and Schuell Co., Keene, N.H. 03431; Glue, Acryloid B-7, Rohm and Haas, Phila- delphia, Pa. 19105). The S&S grade B-12 filters are well suited to diffusion cbam- ber construction because they are flexible, thin (120 q) and impermeable to cells (0.15 pM pore size)l. But they are not so useful as a substratum for growing cells if morphological study is anticipated; they deteriorate in ethanol.

All Millipore filters were boiled for 5 minutes in each of three changes of a large volume of distilled water, dried between filter papers, and stored until used for chambers. S&S filters were used directly from the package.

Single chambers were made from a single ring of polycarbonate plastic 20 mm outside diameter, 10 mm inside diameter and 2 mm thick. A chamber, 2 mm deep was formed when S&S B-12 filters were sealed to the ring with a circle of plastic adhesive tape. (Tapes were pre-cut to size, 19 mm outside diameter, 10 mm inside diameter, by Mr. James Haist, c/o W. B. Kilhour, 25th & Reed Sts., Philadelphia, Pa., 19146); (Bartlett and Prehn, 1969). At the end of the experi-

1 For the record and contrary to a previous report, (Nettesheim, et al. 1966, p. 430), we have always used cell-impermeable S&S filters as the outside filters on diffusion chambers ( SheJton and Rice, 1958).

222 SHELTON, BINNED AND ORENSTEI~

ruent the tape and filter were peeled off and the rings were washed clean and used again.

Both types of chamber as well as the top Hters affixed to the plastic adhesive tape were sterilized and stored in Petri dishes wrapped in paper. Chambers were sterilized either with one side open, to permit introduction of cells, or com- pletely assembled, if they were to be conditioned by implantation into mice. Double chambers were sterilized by dry heat (48 hr at 78°C). Single chambers closed with filters attached to tape, were sterilized by ethyIene oxide gas (Sterox- 0-Matic, W&not Castle Co., Rochester, N.Y.) because at temperature over 40°C the adhesive material in the sealing tape melted and spread into the pores of the filter and so blocked the passage of fluid.

Conditioning of Chambers. We found, as did Goodman et al. (1972), that cell survival was improved when the chambers were in some way “conditioned” before cells were introduced. In t;ivo conditionil~g was achieved by implanting chambers intraperitonealIy into mice (two chambers per mouse) and ahowing them to remain for 4-7 days. At the time of an experiment, they were removed, wiped clean with sterile gauze sponges and placed on a plastic block surrounded with saline-soaked cotton in a Petri dish. The tops were removed to expose the &rid-filled compartment into which the cells were to be put. Excess fluid was pipetted off before the cultures were prepared.

In vitro conditioning was a simpler, less expensive and less time-consuming method. A holder was made from a disc of $in. thick polycarbonate plastic in which were bored a series of shallow holes to receive the chambers. The holes were made in such a way that the plastic ring of the chamber could sit on a ledge while a well in the center beneath the chamber could be filled with fluid. The holder was surrounded with cotton and sterilized in a Petri dish. For con- ditioning, the cotton was soaked in saline, the wells were filled with PBS or MEM and the chambers were set in place. A drop of fluid was put inside each chamber to ensure that the bottom filter was thoroughIy wetted, and the dish was put into a moist 37°C incubator for 30 min before the cells were introduced.

Preparation of Cultures. The cells were grown on separate discs of Millipore filter, 6 mm in diameter (punched from larger filters) with 0.45 ,.&I or 0.025 pM pore size. These “floating filters” were placed in the conditioned chambers and allowed to become wet from below. Lang-Levy Pipettes (3, 5, or 10 ~1) were used to place droplets of fluid containing known numbers of cells in the middle of the floating filters. As long as the top surface of the floating filter was not wetted directIy but aIlowed to become wet from below, the fluid remained as a discrete drop and the cells settled and adhered in a small circumscribed area ( Shelton and Orenstein, 1975).

Cells. Peritoneal cells were collected under sterile conditions. One mouse provided celts sufficient for an experiment. The mouse was shaved on the ventral surface, wiped clean with 70% ethanol and under ether anesthesia was ex- sanguinated by cutting the subclavian artery. The blood was collected with a Pasteur pipette, pooled in a plastic tube and the serum used to determine the antibody titer. The exsanguinated mouse was immediately injected intraperi- toneally with 0.5 ml or 1.0 ml Dulbecco’s phosphate buffered saline without Ca?+ and Mg” (PBS) or Eagle’s minimum essentiaf mediuln (ME-M) and rotated gentfy to distribute the Auid evenly throughout the viscera. The skin was reflected

CELL DIF~~NTIATION IN DIFFUSION CAMBERS 223

and the fluid, amounting to 0.3-0X3 ml, was withdrawn with a Pasteur pipette through a small hole cut in the abdominal muscle. The cell suspension thus ob- tained was stored on ice in a plastic tube. The cell concentration was determined (Celloscope, Particle Data, Inc., Ehnhurst, Ill. ) and aliquots of the fluid contaiu- ing known numbers of cells were mixed with the antigens before the cells were pipetted into the chambers,

Antigens. Sheep erythrocytes (SRBC) obtained fresh weekly and stored in sterile Alsever’s sohrtion, were washed three times in sterile normal saline and made to a concen~ation such that when diluted in the peritonea1 cell suspension the ratio of SRBC to peritoneal cells was appro~mately 1: 1. Typically, 5 ~1 of SRBC suspension was. added to 0.1 ml cell suspension.

Horseradish peroxidase, Type VI, (Sigma Chemical Co., St. Louis, MO.) was dissolved in normal saline at a concentration of 7 mg/ml, filtered through a Millipore filter, 0.45 ,.LN pore size, and stored frozen, At the time of an experi- ment, one part of HRP solution was mixed with 10 parts of sterile normal mouse serum (NMS-HRP) or with sterile immune serum at a hemagglutinin titer log2 = 9 (OPS-HRP) and allowed to sit on ice for 1 hr. One part of one of these mixtures was then added to 10 parts of the peritoneal cell suspension just before the cells were added to the chambers.

Donor Mice. Strain AAFl (A/He X AL/N) female mice between 5 and 9 months of age were used as donors of normal and sensitized peritoneal cells. At 2 to 4 months of age, batches of mice were immunized by injection into the tail vein of 2 x lo* washed SRBC in 0.5 ml saline or by subcutaneous injection in the interscapular region with 250 or 500 rgm of HRP in 0.25 ml complete Freund’s adjuvent (Difco Lab., Detroit, Mich.). Development of antibody titers was followed by direct and indirect hemagglutination with microtiter method (Sever, 1962). Both antigens elicited titers of 6-12 log, within 30 days; titers remained at those levels for up to 6 months. Un-immunized mice of the same age were set ‘aside for use as donors of “normal” cells.

Ho& mice. Mice between 11 and 13 months of age were used for ~pI~tation of diffusion chambers by laparotomy. In most instances, they were AAFl females, but in some of the early experiments, C&HfB/He females were used.

It was found from experience that when there was bleeding into the peritoneal space of the host animal a fibrin clot developed in the chambers. Therefore, great care was taken to avoid trauma to the mice during preparation for and performance of the surgical procedures. The ventral surface of the mouse was shaved and an intraperitoneal injection of sodium pcnabarbital in 10% ethanol (dose = 0.01 ml/gm of 1 mg/ml) provided anesthesia lasting 45 min and deep enough for surgery. During anesthesia and recovery from surgery the animah were kept warm. Lap~otom~es, through a 2 cm incision in the midline, were performed as cleanly and sterily as possible. After insertion of a chamber, muscle and skin were closed separately with three or five interrupted sutures of 6-O silk attached to a fine, straight sewing needle.

Histology. When chambers were to be recovered, the mouse was killed by exsanguination under ether anesthesia; the chamber was removed, wiped clean, and opened; and the fluid was removed with a pipette and rubber bulb to a plastic test tube and saved. The floating filter was fixed in 2% glutaraldehyde in 0.097 M cacodylate buffer, pH 7.4 and stained in toluidine blue according to

224 SHELTON, BENNETT AND ORENSTEIN

a method described previously (Shelton and Orenstein, 1975) with the following variations:

SRBC chambers: After fixation and rinsing in buffer, sheep erythrocytes were stained with diaminobenzidine (DAB). Filters were immersed for 10 min at room temperature in 10 ml 0.05% M Tris-HCI buffer, pH 7.4, containing 3 mg of 3,3’-diaminobenzidine tetrahydrochloride (Sigma Chem. Co., St. Louis, MO.) and 0.010/o H202. They were then stained and mounted on slides.

HRP chambers: Anti-HRP antibody was visualized by a slight modification of the method of Leduc et al. (1968). Floating filters were fixed for 1530 min in 1.25% glutaraldehyde on 0.067 M cacodylate buffer, pH 7.4, containing 1% sucrose, rinsed three times (5 min each) in 0.05 M Tris-HCL, pH 7.4, incubated in Tris buffer containing 0.05 mg/ml HRP (Type VI, Sigma Chemical Co., St. Louis, MO.) for 1 hr at room temperature, rinsed three times (5 min each) in normal saline and incubated in DAB as above for 10 min with constant, slow agitation in a 37°C water bath. After three rinses in normal saline, the filters were stained in toluidine blue and mounted on slides.

The metachromasia of the toluidine blue was extremely useful in following the differentiation of the peritoneal cells and it permitted the identification of plasmablasts whose smooth basophilic cytoplasm was readily distinguished from the pale, delicately fenestrated cytoplasm of the fibroblast.

Light photomicrographs were taken on 35 mm Panatomic X film with a Zeiss Photomicroscope II.

RESULTS

The number of days after which antibody could first be detected in response to either antigen was related inversely to the number of peritoneal cells intro- duced into the chambers (Tables I and II). -However, it was a general feature

TABLE I

Summary HRP Chambers

Exp. no.

Cell donor No. cells Antigen No. chambers with Ab secreting cells/ into No. chambers examined

chamber Days Day 6 Day 8 Days

Days Donor x10-4 I-3 lo-11 after serum

primary titer; immuni- log,

zation

1 82 8 19

2 172 8 14 3 160 8 12 4 89 9 8

5 188 7 2.5

NM@ OPS OPS OPS NMS OPS

OPS

o/2 2/2 2/2 o/2 2/2 263 o/2 o/4 3/3 o/g z/3 O/4 o/2 o/4 l/2

o/2 o/2 l/2

0 NMS = Antigen mixed with normal mouse serum; OPS = antigen mixed with immune mouse serum. See Materials and Methods.

TABL

E II

S~~S

~C

cham

bers

- 63

BP

. ca

ll do

nor

No.

ce

lls i

nto

H~~g

luti~

tit

er

iogt

at

:

no.

cham

ber

X 10

-c

dw

F u

Day

s af

ter

Dono

r se

rum

P

SRBC

l-5

0

8 10

12

20

25

-35

45-5

5 67

pr

imar

y tit

er;

loge

ce

lls

~

imm

uniza

tion

3 1

68

12

9 no

ne

ot

0 0

0 E;

9

9 5;

6

1x;

12

1.1;

14

12

; 15

2

41

12

7 no

ne

0;o

0;o

i2

7 16

0;

o 5;

5

3@

82;

169

10;

10

6 no

ne

0 (4

) 0

0;

0 2

6 10

a

(6)

0;0;

6 3

12;

12

c2

4 77

8

1.2

2 0;

10

; 10

G

10

; 12

z

2.5

4 14

; 12

5

8 0;

Xl;

13

5!

10

16

12;

13

5 13

5 11

8

8 0

7;

1221

2 0;

3;

12

3; 1

3 6b

58

; 96

; 90

; 12

; 9;

7;

2.44

2.

4?5

0 (2

0)

4;

5 0

(6);4

;4

if!

141;

I.6

1 6;

8 8;

8;

IO;

11;

II.

B

0 Su

mm

ary

of tw

o ex

perim

ents

. b S

umm

ary

of f

ive

exp&

men

ts.

Num

bers

in

par

enth

eses

eq

ual

num

ber

of c

ham

bers

. 4 T

iter

of in

divid

ual

cham

ber.

226 S~LTON, BENNETT AND ORENST~IN

FIG. 1. Horseradish peroxidase when mixed immune complex (OPS-HRP) is visible after

with sensitized peritoneal cells as a soluble 24 hr of culture. Note the black diamino-

benzidine reaction product in many lymphocytes and macrophages. x1053. FIG. 2. Horseradish peroxidase incubated with normal mouse serum (NMS-HRP) and added

to sensitized peritoneal cells is absent from most cells after 24 hr of culture. ~1053. FIG. 3. Various stages of antibody secretion can be seen in this group of plasma cells.

g-day chamber; OPS-HRP. X504.

CELL DIFFERENTIATION IN DIFFUSION CHAMBERS 227

of the chamber cultures, regardless of inoculum size or antigen, that the first 3 days were marked by cell death. The severity of this attrition varied from chamber to chamber within an experiment and the presence or absence of plasma cell differentiation could be directly related to cell survival.

Response to HRP us Antigen: The filters from every chamber (Table 1) were processed through the Leduc-Avrameas procedure (Leduc et al. 1968); thus, it was possible to follow the uptake and retention of antigen during the first 3 days of culture and to detect antibody within plasma cells after their differentia- tion at later times. When soluble ~tig~-antibody complexes (OPS-HRP) were mixed with sensitized cells, antigen could be detected in many of the lympho- cytes and in all of the macrophages at 24 hr (Fig. 1). After 3 days, antigen was still present in most macrophages and in roughly 20% of the lymphocytes. By contrast, antigen mixed with normal mouse serum (NMS-HRP) was visible in very few l~phocytes and in about one-half of the macropbages at 24 hr (Fig. 2). At 3 days, NMS-HRP could be detected only in an occasional lymphocyte and in roughly 10% of the macrophages.

During the first 3 days of culture the cells showed no overt sign of differentia- tion in response to HRP. There was no obvious increase in the number of mitoses above that expected for peritoneal cells in chamber culture (Shelton and Rice, 1959) and no increase in the size or basophilia of the lymph~ytes. However, at 6 days or later, depending on inoculum size, plasma cells stained for anti-HRP antibody were detected in each experiment.

In chambers where specific differentiation had occurred, some plasma cells “fully committed” to the secretion of anti-HRP antibody were completely black- ened by the DAB reaction product, in others, the reaction product was seen to occupy only part of the cytoplasm and leave the remainder totally unstained (Fig. 3). These cells were readily detected and did not exceed 10% of the total plasma cell population in any chamber. On the other hand, in fully 50% of the plasma cells no anti-HRP antibody could be detected.

That antibody was present in the remaining plasma cells was established only by close scrutiny of each cell. The preservation of the entire culture as a “whole- mount” made it possible to “dissect” a cell from top to bottom by focusing with an oil immersion lens and, in so doing, many cells were found to have small areas that were clearly stained with the DAB reaction product. These areas were found in patches associated with the cell surface or in patches in the perinuclear space of the endoplasmic reticulum (Figs. 4,5,6). Such small, isolated areas were not usually found in regions of the cytoplasm intermediate between perinuclear space and plasma membrane.

No antibody titer (by indirect hemagglutination) could be detected in fluid from chambers containing plasma cells stained positively for anti-HRP antibody.

FIG. 4. The variability in localization of specific anti-HRP antibody is clearly displayed by these clusters of plasma cells from an g-day chamber (OPS-IIRP). In Fig. 4, patches of specific antibody can be seen in the perinuclear space and at the periphery of the cell ( arrows). ~918.

FIG. 5. One cell is lightly stained throughout (arrow). X 1229. FIG. 6, The localization of anti-HRP antibody in only part of the entire endoplasmic retic-

ulum of these cells graphically conveys the ~p~sion of a cycle of antibody secretion. S-day chamber; OPS-HBP. X 1494.

228 SffELTON, BENNETT AND O~NSTEIN

9

FIG. 7. Primed peritoneal cells exposed to SRBC and cultured for 24 hr. Three plasmablasts with large pale nuclei and prominent nucleoli can be identified among small and large lymphocytes. The complete flattening of the cells on the filter exaggerates their size. x801.

FIG. 8. Clusters of plasmablasts surround macrophages (arrows) in this 72 hr culture of primed peritoneal cells incubated with SRBC. Large, dark cells are mast cells. ~347.

FIG. 9. The alteration in morphology of the lymphocyte membrane illustrated here was an- other typical occurrence in 72 hr cultures of primed cells incubated with SRBC. Compare

CELL DIFFEZBENTIATION IN DIFFUSION CHAMBERS 229

Response to SRBC as Antigen. Dtierentiation of peritoneal cells into antibody secreting plasma cells after exposure to sheep erythrocytes (SRBC) was assessed by measuring the hemagglutinin titer of the chamber fluid. No measurable hemagglut~a~g antibody was present in chambers where cells from unimmu- nized mice plus SRBC or cells from ~immun~ed mice plus SRBC or cells from immunized mice minus SRBC were cultured. When primed cells were mixed with SRBC, hemagglutinatiug antibody was first detected after 6 days of culture (Table II), and after that time, high antibody titers were recorded. Plasma cells and high antibody titers continued to be observed in chambers for up to 67 days of culture (Experiment 5, Table II). In most cases the titers were positively correlated with the presence and number of plasma cells in the cham- bers, but there were notable exceptions to this rule. The few cases were a modest number of plasma cells was matched with a high antibody titer could be ex- plained on the basis of cell loss, but the reverse was Iess easily dismissed. In two cases, chambers contained large numbers of plasma cells but had no de- tectable antibody in the chamber fluid although high titers were recorded in other chambers of the same group.

At 24 hr, slender cytoplasmic processes making contact between lymphocytes were frequently seen in cultures of both normal and sensitized cells, but the striking rno~hoIo~~~ d~erentiation that developed in sensitized peritoneal cells after exposure to SRBC did not occur in normal ceils exposed to SRBC or in sensitized celIs cultured in the absence of SRBC. Cultures of sensitized cells displayed mitotic figures and cells whose large, pale nuclei and prominent nucleoli were surrounded with generous basophilic cytoplasm ( Fig. 7). These were typical pIasmablasts. At 72 hr, two further changes in the culture occurred. First, there appeared large clusters of basophilic cells, many in mitosis, and usually sur- rounding a macrophage (Fig. 8). Second, there was a change in the shape of many otherwise undifferentiated Iymphocytes. The cytopfasm of these cells be- came spread out around a flattened, elongated nucleus in diverse patterns of sheets and strands that imparted to the fixed preparations an ‘aspect of frenetic activity (Figs. 9, 10). These alterations were repeatedly observed in different experiments.

Differentiation continued with time, and great numbers of plasma cells ap- peared. In older cultures these were debuted over a monolayer of macrophages or ~broblasts that covered the filter surface. Clusters of plasma cells around macrophages were always seen, however, not all cells were involved and it was frequently possible to find lymphocytes whose morphology was unaltered even when other lymphocytes nearby were differentiating (Fig. 9).

Contacts between cells were observed repeatedly and most strikingly between plasmablasts and macrophages at 48 and 72 hr, or between fully differentiated plasma cells and macrophages at later times. Extendiug from many of these basoph~c cells, which were disposed singly or in clusters beside or above a macrophage, couId be seen thick cytoplasmic processes or broad flaps of cyto- pIasm which made contact with the macrophage surface (Figs. 11, 13, 14).

with Fig. 8. The large, dark cells are mast cells. Fig. 9, X488. FIG. 10. X480. FIG. Il. At 72 hr of culture, overt interaction between lymphocytes plasmablasts and macrophages was com- monly observed. Cytoplasmic processes extended from the plasmablasts to the mesothelial cells and macrophages. Fig. 11, X1020.

230 SULTAN, BENNE~ AND O~N~TEIN

FIG. 12. Interaction between mature plasma cells and macrophages or sheets of mesothelial cells were common events in older cultures of primed peritoneal ~11s exposed to SRBC. In Fig. 12, individual plasma cells extend processes to make contact with a macrophage. &day culture, x640. At later times, the plasma cells formed clusters over the macrophages and mesothelial cells. Fm. 13. X1560. FIG. 14. X640. FIG. 15. 11 days, X640. FIG. 16. 35 days, X365.

CELL DIFFElRENTIATION IN DIFFUSION CHAMBERS 231

Occasionalfy, the tip of the process was disposed in a bouquet of ruffles over the surface of the ma~rophage (Fig. 13). Plasma cells from 6 days onward were seen to cluster around and above ma~rophag~ in preference to the filter surface and close contact with the macrophage surface could frequendy be dist~guished (Figs. 12, 15, 16).

DISCUSSION

Mouse peritoneal cells, when cultured in diffusion chambers and challenged with antigens as diverse as sheep erythrocytes and horseradish peroxidase, pro- vide a model system of the immune response. This system is suitable for study of cytologic changes associated with differentiation, rates and sites of antibody synthesis, and interactions between cells of different types. In the absence of a flux of cells from exogenous sources, it is possible to look at popula~ons of plasma cells that are progeny derived from a restricted pod of precursor cells. In the present experiments, it was found that, in terms of the appearance and intracellular localization of antibody and the percentage of cells secreting specific antibody, the differentiation of these cells proceded in accordance with data derived from the study of antigen stimulated lymph nodes (Leduc et al. 1968; Avrameas et al. 1967; Murphy et al. 1972; Hay et al. 1972; Miller et al. 1973a, b). The immune response to HRP has been studied principally in spleen and lymph nodes of rabbits, mice, rats, and sheep and the results of these studies have been summarized recently in papers by Murphy et ab ( 1972), Hay et aI. ( 1972) and Miller et al. (1973a; 1973b). The differentiation of peritoneal cells in diffusion chambers has paralleled and extended the ~formation derived from these in o&o experiments.

The number of plasma cells observed in the HRP cultures, while substantial, was never comparable to that seen in the SRBC chambers. The response of primed peritonea1 ceIIs to sheep erythrocytes was overt, rapid and powerful. Large numbers of plasma cells accompanied by the appearance of substantia1 hemagglutinating antibody titers, were generated within 6 days. Plasma cells and antibody titers persisted for over 2 months in chambers receiving an initial inoculum of 80,000 cells (Table II) Nettesheim et al. (1966) by using a larger chamber inoculum of 25 X 10” primed spleen cells were able to detect antibody titers after four months of culture.

Two features of the response to sheep erythrocytes deserve further clarification by future expe~ments. The first is the rem~kable alteration after 48 hr in shape but not in size of the lymphocyte. The alteration in both nuclear and cytoplasmic membranes imparted the strong impression of movement to the individual cell and to the culture as a whole. Not all lymphocytes were so affected and many could be seen to retain their characteristic rounded shape. This type of mor- phologic alteration was not seen in cultures of unpruned peritoneal cells ex- posed to sheep erythrocytes. Similar changes in the structures of membranes of lymphocytes within lymph nodes are illustrated in a report by J. And&-Schwartz (1964; Figs. 2 & 3) who described the ultrastructure of cells involved in graft rejection. In control lymph nodes from normal rabbits, the surface of lympho- cytes was smooth in outline, but at the height of first-set graft rejection (7 days), tbe membranes of lymphocytes in the draining lymph nodes were extended, convoluted and interdigitated.

232 SHELTON, BENNETT AND ORENSTEIN

T&e second feature of the response to SREC in the chambers was the intcr- action of plasmabl~ts and plrasma cells with macrophages, From 48 hr to 67 days the pIasmablast and then the plasma cells were found in very close association with the n~a~rophage, Cell processes, slender anes and stout ones, extended from these cells to make contact with the macrophage surface, That lymphocytes make contact with macrophages by means of processes has been thoroughly described and documented by Berman ( 1966). His time-laspse photographs of cultured human peripheral blood lymphocytes and macrophages with and without phyto- hemagglutinin showed that lymphocyte pseudopods extended and retracted, changed in width ,and length, and made contact with and even occasionally in- dented the surface of macrophages. More recently, McFarland and Sheeter ( 1970) have suggested that microspikes on the “uropod” of the lymphocyte are part of the ‘Surveillance” function of the l~phocytes.

In the present experiments, rather than the lymphocyte, it was the ~Iasmablast early in the immune response or the mature plasma cell late in the response that interacted with other cells. It might be hypothesized that the plasmablast was receiving a “message” to differentiate, but, presumably, the plasma cell was already committed to the production and secretion of immuno~lobulin and thus would not be in need of such a stimulus.

The propensity of plasma cells in the chambers to cluster around the macro- phages and mesothelial cells bears upon two other phenon~ena; the infrequent appearance of plasma cells in the peripherial circuIation of immunized animals and the requirement of a feeder layer for in vitro propagation of plasma cell tumors, It is known that very few plasma celb escape the lymph node in the efferent lymph after the node has received antigen via the a&rent lyn~phati~ (gushy et al. 1972) and it appears likely from our observations that the pIasma eelIs couId form a strong enough association with the reticular celIs of the node to prevent their entering the lyn~pha~i~ vessels. In regard to the second goint, success in propagating murine and human myelomas in cuhure appears to depend upon the presence of feeder layers or ancillary growth factors and recently Jabin et al. (1974) have pointed out that the dependence of the malignant pIasma cell upon growth-supporting factors derived from norma cells may be important for an understanding of multiple myeloma.

In contrast to the response to sheep erythrocytes, the response of peritoneal cells to the HRP molecules was a very subtle one. So far as the present experi- ments went, progress to the plasma cell was accomplished without striking aftera- Cons in the general ~chite~~re of the cultures, However, there are gaps in our observations of the RR? response: no chambers were examined at 4 or 5 days, and future study should provide information about this interval. Among the hundreds of fully differentiated plasma cells present in the chambers from 6 days onward, only a few were fully committed to the production of anti-HRP antibody as indicated by intense blackening with DAB reaction product. In many cehs, small amounts of reaction product were present either in the perinuclear space or in patches on the plasma membrane or on rare occasions in both loca- tions in the same cell. This observation con&ms those of Ledue and others (Leduc et al. 1968; Miller st uZ. 1973a) and strengthens the interpretation that the synthesis of specific antibody by plasma cells occurs in a discontinuous cycle that starts at the nudeus and progresses to the cell membrane where antibody

CELL DI~FE~NT~TI~N IN DIFFUSION CHAMBERS 233

is secreted. It is not clear, however, that the uhimate secretion is accomplished by pinching-off of fragments of cytoplasm (Leduc et al. 1968) since this phe- nomenon was only occasionally observed,

It was further apparent that in chambers where many plasma cells were syn- thesizing anti-HRP antibody, there was equally large numbers in which no spe- cific antibody could be demonstrated. Since Humphrey (1963) first reported that nonspecific gamma globulin is formed in response to Freund’s adjuvent, evidence has accumulated that plasma cells synthesizing non-specific immunoglobulin formed in response to such antigens as diptheria toxoid (Balfour et al. 1965) tobacco virus ( Urbana-Vansanten, 1970)) and horseradish peroxidase Avrameas and Leduc, 1970). Recently, Miller et aI. ( 1974) have presented evidence that during the immune response to HRP, both specific antibody and non-specific immunoglobulin can be detected within the same cell. They also find support for the idea that plasma cells secreting specific antibody may be recruited from a pool of cells synthesizing nonspecific immunoglobulin. These results may indicate that although ~munoglobulin is synthesized by all plasma cells, the specific antigen-bind~g sites of the molecule are intermittently expressed or not ex- pressed at all.

ACKNOWLEDGMENT

The skilled technical assistance of Mrs. Ann Soria, Mr. Charles Mock and Mr. W. Mowczko is gratefully a~owledged.

REFERENCES

ANDRE-SCHWARTZ, J. ( 1964). The morphologic responses of the lymphoid system to homo- grafts. BEood 24, 113-133.

AVRAMEAS, S., and LEDUC, E. H. (1970). Detection of sirnult~~~ antibody synthesis in plasma cells and specialized lymphocytes in rabbit lymph nodes. J. Exp. Med. 131, 1137- 1168.

A.VRAMEAS, S., and LESPINATS, G. (1967). Detection d’anticorps dans les cellules immuno- competentes d’animaux immunises avec des enzymes. C. R. Acad. Sci. Ser. (B) (Pa&s) 265, 302-304.

BALFOUR, B. M., COOPER, E. II., and ALPEN, E. L. ( 1965). Morphological and kinetic studies on antib~y-producing cells in rat lymph nodes. Zmrn~~~g~ 8, 230-244.

BARTLETT, G. L., and PREEN, R. T. (1969). AR improved design for genera1 purpose diffusion chambers. Transplantation 7, 225-227.

BERMAN, L. ( 1965). Lymphocytes and macrophages in uitro. Lab. Invest. 15, 1084-1099. BORELLA, L. ( 1971). Regulation of IgM memory expression by spleen cells cultured in

diffusion chambers. Immunology, 26, 289-298. BOYUM, A., and BORGSTROM, R. ( 1970). The concentration of granulocytic stem cells in mouse

bone marrow determined with diffusion chamber technique. Stand. f. ~~~. 7, 294303. CAPALBO, E. E., ALBRIGET, J. F., and BENNETT, W. E. (1964). Evaluation of the diffusion

chamber culture technique for study of the morphological and functional characteristics of lymphoid cells during antibody production. J. Immunol. 92, 243-251.

COOPER, M. D., LAWON, A. It., and KINCADE, P. W. ( 1972). A two-stage model for develop- ment of antibody-producing cells. C&n. Exp. Immunol. 11, 143-149.

GOODMAN, S. A., M. G., and MAKINOD~, T. ( 1972). An improved primary response from mouse spleen cells cuhured in o&o in diffusion chambers. I. Zrnrn~~Z. 168, 1387-1399.

by, J- B., MURPHY, M. J., Moxms, B., and BESSIS, M. C. (1972). Quantitative studies on the proliferation and differentiation of antibody-forming cells in lymph. Amer. J. Path. 66, l-17.

Honr.m, M. ( 1960). Morphology and antibody production by diibxent cell systems in diffusion chambers. Folia Microbial. 5, 347-362.

SHELTON, BENNE~ AND O~ENSTEIN

Hom~n, M., and RmA, I. ( 1960). ~~orphologi~l changes in lymphocytes cultivated in diffusion chambers during primary response to a protein antigen. In “Mechanisms of Antibody Forma- tion.” Holub and Jaroskova (Eds.), pp. 30-35.

HuMPmu% J. H. (1963). The non-spec&c globulin response to Freud’s adjuvent. CoZZuq. Centre Nat. Rech. Sci. 116, 401409.

JonIN, M. E., FAHEY, J. L., and PRICE, Z. ( 1974). Long-term establishment of a human plasmacyte cell line derived from a patient with IgD multiple myeloma. J. ~xp. Med. 140, 494507.

Lnnuo, E., AVRAMEAS, S., and BOUTEILLE, M. (1968). Ultrastructural Iocalization of antibody in differentiating plasma cells. J. Exp. Med. 127, IOQ-118.

MAKINODAN, T., HOPPO, I., SADO, T., CAPALBO, E. E., and LEONARD, M. R. (1965). The suppressive effect of supraoptimum doses of antigen on the secondary antibody-fo~~g response of spleen eelIs cuhured in ceIi-impernleable diffusion chambers. J. Immuno~. 95, 466479.

MUINODAN, T., NETTESEIEIM, P., h4ORITA, T., and CHADWICK, C. J. ( 1967). Synthesis of antibody by spleen cells after exposure to kiloroentgen doses of ionizing radiation. J. Cell Physiol. 69, 355-366.

MCFARLAND, W., and SCHECTER, G. P. ( 1970). The lymphocyte in immunological reactions in uitro: Ultrastructural studies. Blood 35, 683-688.

h4ILLER, H. R. P., AWIAMEAS, S., and TERNYNCK, T. (1973a). Intracellular distribution of antibody in immunocytes responding to primary challenge with horseradish peroxidase. Amer. J. Path. ‘71, 239-255.

MILLER, H. R. P., AVRA~IEAS, S., and TERNYNCK, T. ( 1973b). Intracellular distribution of antibody in immunocytes responding to secondary challenge with horseradish peroxidase. Amer. J. Path. 71, 261-273.

MILLER, H. R. P., TERNYNCK, T., and AVRAWEAS, S. (1974). A comparison of specific antibody and immunoglobulin synthesizing ~muno~ytes in peroxidase stimulated lymph nodes. Ann. Imrn~~nol. (Irrsst, Pastew) 125C, 231-238.

MILLER, J. F. A. P. (1972), Lymphocyte interactions in Antibody Responses. ht. Rw. Cytol. 33, 77-130.

MURPHY, M. J., HAY, J. B., MORRIS, B., and BESSIS, M. C. (1072). Ultrastructural analysis of antibody synthesis in cells from lymph and lymph nodes. Amer. J. Path. 66, 25-35.

NETTESHEIM, P., and MAKINODAN, T. ( 1965). Differentiation of lymphocytes undergoing an immune response in diffusion chambers. J. Immunol. 94, 868-870.

NETTESHEI~I, P., MAKISODAN, T., and CHAD’WICK, C. J. ( 1966). Improved diffusion chamber cultures for cytokinetic analysis of antibody response. Immunol. 11, 427439.

NOSSAL, G. J. V. ( 1971). Recent advances in immunological tolerance. In ‘Progress in Im- munology.” B. Amos (Ed.), pp. 665-677.

NOSSAL, G. J. V., and ADA, G. L. (1971). “Antigens, Lymphoid Cells, and the Immune

Response,” Academic Press, New York. PREHN, R. T., WEARER, J. M., and Algire, G. H. (1954). The diffusion chamber technique

applied to a study of the nature of homograft resistance. J. Nut. Cancer Inst. 15, 509-516. ROSENSTRIXCK, D. I..., BLAKE, J. T., and ROSENTHAL, A. S. (1971). The peritonal exudate

lymphocyte. I. Differences in antigen responsiveness between peritoneal exudates and lymph node lymphocytes from immunized guinea pigs. J. Exp. Med. 134, 1170-1186.

SEGR$ M., and SEC&, D. ( 1972). Anti-DNP hemolytic plaques by mouse spleen cells in diffusion chambers. lmmunol. Comm. 1, 143-153.

SEVER, J. L. (1962). Applications of a microtechnique to viral serological investigations. ]. Immunol. 88, 320329.

SHELTON, E., DAVES, S., and HE~LIIIIER, R. ( 1970). Q uantitation of Strain BALB/c mouse peritoneal cells. Sc+e:ience 168, 1232-34.

SHELTON, E., and RICE, M. E. (1959). Growth of norma peritoneal celis in diffusion cham- bers: A study in cell modulation. Amer. J. Anat, 105, 281-342.

SHELTON, E., and RICE, M. E. (1958). Studies on mouse lymphomas. 11. Behavior of three lymphomas in diffusion chambers in relation to their invasive capacity in the host. J. Nat. Cancer ht. 21, 137-161.

SHELTON, E., and ORENSTEIN, J. M. (197s). A plating method for preparation of cells for culture and for observation by light or electron microscopy. Exp. Mol. Path. 23, 226-227.

CELL DIF~~NTIATION IN DIFFUSION CHAMBERS 235

URBAIN-VANSANTEN, G. ( 19’70). Concomitant synthesis in separate cells of non-reactive im- munoglobulin and specific antibodies after immunization with tobacco mosaic virus. Zm- munology 19, 783-797.

VANN, D. C. ( 1969). In Vitro antibody synthesis by diffusion chamber cultures of spleen ceils. II. Effect of increased levels of free antibody. 1. ZmmunoZ. 102, 451456.

VANN, D. C., and MAXINODAN, T. (1969). in V&o synthesis by d&&on chamber cultures of spleen cells. I. Methods and effects of 10,COO r on antibody synthesis. J. Zmm~nol. 102, 442-450.

WEILER, I. J. ( 1965). Antibody production by determined cells in diffusion chambers. J. Zmmunol. 94, 91-98.