Growth of normal peritoneal cells in diffusion chambers: A study in cell modulation

GROWTH O F NORMAL PERITOXEAL CELLS I N DIFFUSION CHANBERS: A STUDY

I N CELL MODULATION

EMMA SHELTON AND MARY E. RICE Laboratory of Biology, National Cancer Institute:

Bethesda, Maryland

SEVENTY-NINE FIGURES

I n recent studies on the growth of lymphocytic ascites tumors in diffusion chambers it was observed that fibers and viscous fluid regularly appeared in chambers where one of the tumors was grown (Shelton and Rice, '58). Periodic examination of the cells in the chambers revealed that the fibers and viscous fluid were not produced by the tumor cells but by the normal peritoneal cells that had been introduced into the chambers together with the tumor cells. This observation led to a study of the morphologic and physio- logic modulations that occur in normal peritoneal cells when they are grown in diffusion chambers. The diffusion chamber technique has thus provided a new approach to the prob- lem of the fate and function of the free cells of the reticulo- endothelial system - particularly the lymphocyte and the macrophage.

EXPERIMENTAL

The experimental procedure employed was as follows : known numbers of the free cells of the serous fluid of the peritoneal cavities of Strain A mice were introduced into diffusion chambers and the chambers were in turn inserted by laparotomy into Strain A mice or Holtxman rats. The

Received for publication, January, 1960. National Institutes of Health, Public Health Service, U. S. Department of

Health, Education, and Welfare. 281

THE A Y E R I C A N JOUILNAL OF ANATOMY, VOL. 105, NO. 3 November 1959

282 EMMA SITELTON AND MARY E. RICE

chambers were allowed to stay in the animals for periods ranging from 18 hours to 145 days, after which the animals were killed by cervical dislocation, the chambers removed and their contents analyzed. A total of 126 chambers were examined.

Four different combinations of cells and host animals were used.

1. Normal mouse peritoneal cells in the mouse. 2. Mouse peritoneal lymphocytes in the mouse. 3. Mouse peritoneal lymphocytes in the rat. 4. Control chambers containing no cells.

Cells

These cells (Normal Cells) were obtained from the peritoneal cavities of Strain A mice of both sexes ranging in age from three months to 11 months by the following method: one milliliter of Earle's saline containing 0.04 pg of heparin was injected intraperitoneally into 8 to 10 mice. After the last animal was injected, the mice were killed at random by cervical dislocation and the fluid aspirated with a pipette and rubber bulb through a hole in the abdominal muscle - the whole procedure taking between 20 and 30 minutes. In order to avoid trauma to the peritoneum, the pipette was inserted only once into each mouse. The fluids were pooled in a sterile centrifuge tube held in crushed ice and after the collection was complete, they were centrifuged at 250 X g for 5 minutes in an Inter- national refrigerated centrifuge. The pellet of cells was resuspended in the supernatant fluid t o a total volume of 0.5 to 0.7 ml. The concentration of cells in this suspension was determined by direct counts in a hemacytometer as previously described (Shelton and Rice, '58). In addition, differential counts on a total of 1,000 cells were made.

Over a period of 15 months, 10 separate groups of chambers containing Normal Cells were put into mice. There were 5 to 20 chambers in each group and each chamber within

Normal perito.lzenZ cells.

STUDY I N CELL MODULATION 283

a group received the same number of cells (range : 150,000 to 4 X lo6) contained in a volume of 0.02 or 0.03 ml of fluid. Each group of chambers was filled from a different pool of cells.

PeritoNeaE lymphocytes. An almost pure suspension of lymphocytes (P-lymphs) was obtained by allowing the macrophages and granulocytes to separate themselves from the peritoneal fluid by attaching to glass wool fiber^.^ Peritoneal fluids obtained as described above were collected in a 15 X 150 mm test tube that contained loosely packed glass wool. The sealed tube was placed on a roller tube rack and rotated in a horizontal position at one revolution every 5 minutes for one hour at room temperature. As the tube rotated the cells were continually percolated through the meshes of the glass wool and when the fluid was decanted at the end of an hour, it contained 95 to 98% lymphocytes with only 245% macrophages, granulocytes and mast cells remaining. The cells were concentrated and counted as above before being put into the chambers.

Fifteen chambers containing P-lymphs (6 to 9 X lo6 cells) were put into mice in three groups. Three chambers containing mouse P-lymphs were put into Holtzman rats. The rat chambers contained 4.5, 3.4, and 2.3 x l oo cells, respectively, and were removed at 7, 14, and 21 days.

Chambers In. t he mouse. Except for control chambers, double diffu-

sion chambers (Shelton and Rice, '58) were used exclusively. Briefly, they consisted of two lucite rings (1 mm thick, 2 ern outside diameter and 1 em inside diameter) between which was cemented a Millipore HA cellulose membrane filter (pore size 0.45 =t 0.02 p). On the outside of the rings were cemented Schleicher and Schuell Very Dense filters (pore size less than 0.1 p). Thus, between the filters, two cylindrical

3Pyrex glass wool for filtration; Corning Glass Company, Corning, New York. ' Millipore Fil ter Corporation, Bedford, Massachusetts.

Carl Schleicher and Schuell Company, Keene, New Hampshire.

284 EMMA SHELTON AND MARY E. RICE

chambers 1 ern in diameter and 1 mm thick were formed. The chambers were fabricated except for the top filter and sterilized by dry heat at 80°C for 45 hours. Cells were pi- petted onto the center filter, the top filter was sealed to the top lucite ring and the chamber inserted into the host animal.

The Very Dense filters allowed fluid from the peritoneal cavity to enter the chambers, but host cells were excluded.

I n the rat. The chambers used in the rat were similar in design but larger than those used in the mouse. The rings used to support the filters in the rat chambers were 2 mm thick, 45 mm outside diameter, 38 mm inside diameter and were cut from lucite tubing. A Millipore PH filter (pore size 0.3 t 0.02 p) was cemented between two rings and two Milli- pore VC filters (pore size 0.1 t 0.008 p) were cemented on the outer surfaces of the rings to complete the double chamber.

Control chambers were put only into mice. The first type was a double chamber used to test the effectiveness of the Very Delzse filters in keeping host cells out of the chambers. These chambers were put into mice empty and opened after 145 days. The center filters of the chambers were fixed, stained, and examined for the presence of cells.

A second type of control chamber was put into mice f o r the purpose of studying the cells that attached themselves to the outside of the filters. These chambers consisted of a single lucite ring to which were attached two HA filters. The implanted empty chambers were removed at 24-hour intervals for from one to 10 days. The two filters were fixed, stained and examined microscopically.

Go.iztroZ chambers.

Fixatiolz and staining After the double chambers were removed from the animals,

the outer Very Dense filters were wiped clean, cut away by circling the rims of the center holes with a sharp knife and


discarded. The fluid contained in the chambers was drained off and either examined with the phase microscope, frozen for later analysis, or discarded. The center filter, still cemented between the lucite rings, was dropped into 3% potas- sium dichromate (pH 6.8) containing 10% formalin. The filters were fixed at room temperature for three hours or overnight at 5°C.

Most of the filters were stained in Lillie’s Allochrome connective tissue stain (periodic acid-Sckiff, followed by hematoxylin and picromethyl blue) (Lillie, ’54). A few were stained with hematoxylin alone or with oil red 0 for lipid. The center filters were carried through routine histologic procedure and were not removed from between the lucite rings until they were ready to be mounted on slides. The resulting preparations were stained “whole-mounts” of the cell growth which gave an extraordinarily lucid picture of cell relationships.

For electron microscopy, the filter held between the lucite rings was inverted over 2% OsO, for 20 minutes, then rinsed in saline and dehydrated in alcohol. Small pieces were em- bedded in methacrylate.

Determination of hydrogyproline

The hydroxyproline content of the chambers was used as an estimate of the amount of collagen produced by the cells. In preparation for analysis, the contents of the chambers were transferred to a test tube containing 2 ml of 0.1N NaOH. After centrifugation, the tissue was resuspended in 2 ml 0.1 N NaOH and allowed t o stand at room temperature for one hour with occasional stirring. The tissue was extracted twice more with 2 ml of 0.1 N NaOH, washed three times in cold distilled water and extracted twice with acetone. The extracted tissue was hydrolyzed 7 hours at 110-112°C in 6 N HC1. The HC1 was removed by vaporization under vacuum, the hydrolysate was dissolved in 0.1 ml buffered 10%


isopropanol, pH 6.2, and stored frozen until ready for analysis .6

The hydroxyproline content of the extracted collagen was determined by the quantitative horizontal paper chromato- graphic method described by Roberts, Kolor and Busek ('58).

RESULTS

Since it was important that the cells introduced into the chambers be identified, careful, repeated study was made of the free cells of the serous fluid of the peritoneal cavity of the Strain A mouse. These cells consisted of 4 distinct types;

TABLE 1

Differential counts representing a total of 2Y,OOO peritoneal cells obtained from 48 normal Strain A mice of bo th sexes, 3-9 months of age'

PER STANDARD DEVIATION CENT

Lymphocytes 77.6 f 5.07 Macrophages 18.4 i- 6.56 Granulocytes 1.2 2 1.5 Mast cells 2.0 i- 1.0 Transitional cells 0.8 f 0.8

'These counts may be compared with those reported by other investigators (4, 5, 6 ) .

the lymphocyte, the macrophage (histiocyte), the granulocyte (neutrophilic and eosinophilic), and the mast cell (plate 1). Occasionally there was also present a transitional cell that possessed in varying degrees some of the attributes of both the lymphocyte and the histiocyte. Neither plasma cells nor mesothelial cells were found in the peritoneal fluid of the normal, untraumatized animal. Differential counts showed that the 5 cell types normally maintained a remarkably consistent proportion t o each other (table l), but the presence of a diffusion chamber in the peritoneal cavity

Several methods for the extraction and hydrolysis of collagen were compared and found to yield comparable resnlts. The methods tested included autoelaving fat-extracted tissue a t 17-20 pounds pressure in sealed tubes for 15 hours and hydrolysis of various extracts a t a variety of times and temperatures.

STUDY IN CELL MODULATION 287

induced changes in the proportions of the cells (table 2) that were similar to those reported for other inert materials (Felix and Dalton, ’55).

The reaction of the host animal to a large foreign body- the chamber - resulted in about one-third of the chambers becoming attached to the liver or mesenteries. The nature of the attachment depended upon the length of time that the chamber had been in the mouse. At 24 hours, the attachment was tenuous and easily broken by gentle manipulation, but with increasing time the attachment became a stronger link of connective tissue. The chamber itself was enclosed in a progressively thicker capsule which eventually required dis- section for release of the chamber.

Growth of cells i m the chambers. I n an attempt to follow the dynamic events that occurred when peritoneal cells were grown in diffusion chambers, chambers were opened at intervals of 18 to 48 hours during a period of two weeks. Six groups of chambers representing G different pools of cells were included in this time period. Additional groups were studied a t various intervals up to 140 days and in each group essentially the same changes were observed. No quantitative estimate was made of cell survival in comparison with the number of cells introduced into the chambers, but there was unquestionably a certain amount of cell death during the first 48 hours in the chamber. The extent of the initial necrosis, estimated by the number of cell “ghosts” seen on the filters, appeared t o depend upon the individual pool of cells o r the group of chambers used. A summary of the chambers upon which the following description is based is presented in table 3.

General pattern of growth. A survey of the stained filters with low magnification of the microscope afforded a general picture of the relationships of the cells t o each other em nzasse. The characteristic appearance of the cells during the first three days was that of even dispersal on the filter surface. Among flattened macrophages and granulocytes, rounded dense lymphocytes were scattered like grains of pepper.

TABLE 2

Dif

fere

ntia

l cou

nts

of p

erito

neal

cel

ls f

rom

mic

e w

ith

cham

bers

ins

erte

d in

per

itone

al

cavi

ty

DAYS A

FT

ER

IN

SE

ET

ION

0

2 7

14

2

1

28

O

P O

HA

MB

ER

32.0

f 1

3.1

25.4

f 12

.7

Lym

phoc

ytes

74

.3

& 5

.99'

28

.4 f 8.

39

28.3

f 8

.02

26.8

t- 11

.5

28.2

f 12

.1

Mac

roph

ages

21

.4 f 5

.45

10.8

f 4.

81

28.5

f 9

.46

36.3

f 10

.7

33.5

f 1

0.4

Gra

nulo

cyte

s 0.

94 f 0

.80

44.9

&

7.0

0 22

.5 f 10

.5

14.3

&

3.

53

14.9

f

6.83

18

.9

-C

4.41

M

ast c

ells

2.

15 f 0

.86

0.06

& 0

.05

0.01

f

0.05

0.

07 f 0

.05

0.18

f

0.13

0.

17 f

0.19

T

rans

itio

nal c

ells

1.

16 C

0.4

8 15

.4 f 4.

81

20.5

2

3.9

22.4

f

4.65

23

.3 f

4.30

24

.1 2

1.80

N

umbe

r of

mic

e 14

14

14

1

4

in

6

ra

00

00

Fig

ures

rep

rese

nt m

ean

per

cent

and

sta

ndar

d de

viat

ion,

TA

BL

E 3

Sum

mar

y of

st

aine

d fil

ters

DAYS I

N M

OU

SE =

1

2 3

4 5

6 7

8 9

10

11

14

1

5

17

20

21

29

42

53

62

1

31

1

40

T

OT

AL

S

Num

ber

stai

ned

11

21

Nor

mal

per

iton

eal c

ells

N

umbe

r st

aine

d 7

6 6

7 2

44

7 4

2 2

3 2

3 3

3 2

41

4 2

15

93

G

ood

grow

th =

77

Poor

gro

wth

= 1

2 N

ogro

wth

=

4

11

1

11

1

2

2 15

Goo

d gr

owth

= 1

3 Po

or g

row

th =

2

Nog

row

th=

0

Num

ber

stai

ned

1

1

1

Goo

d er

owth

= 3

Peri

tone

al ly

mph

ocyt

es i

n m

ouse

Mou

se p

erit

onea

l lym

phoc

ytes

in r

at

M z P 0

#

Con

trol

cha

mbe

rs:

Dou

ble,

a t

otal

of

14 c

ente

r fi

lters

exa

min

ed a

fter

145

day

s;

Sing

le,

a to

tal

of

26 f

ilter

s ex

amin

ed;

4 ea

ch o

n da

ys 1

, 2,

3, 6

, an

d 8

; 2

each

on

days

4,

5, a

nd 1

0.


There was very little evidence of organized growth. The contrast between the chambers containing P-lymphs and those containing Normal Cells was impressive (figs. 11 and 12), for the macrophages which formed a sizeable proportion of the population in the Normal Cell chambers were barely recognizable in the P-lymph chambers. After a few days, however, the macrophages in the P-lymph chambers were more obvious because of the cloud of PAS-positive material that surrounded them (fig. 13).

After three days, there occurred a dramatic and rapid organization of the cells from their previous condition of separateness to one of colonization. Between 5 and 10 days, there was established what might be termed the “mature pattern of growth” which was characterized by cell sheets or colonies of widely varying size and density, surrounded by a generous sprinkling of discrete, macrophage-like cells (figs. 14, 15, 16, 17). If the organized cell sheet did not fill the whole chamber (it frequently did by 140 days), then invari- ably it was surrounded by unorganized, randomly oriented cells (fig. 67). The sheets of cells were not simply layers of similar cells joined together and piled one on top the other; they were a more complex organization of several types of cells. The basic structural units of the sheets were flattened fibroblasts (figs. 41, 42, 69) held together by crisscrossed layers of collagen fibers (fig. 43), but accumulated at the surface of the sheets were lymphocytes, granulocytes, mast cells, plasma cells and cells in various stages of modulation. These free cells were seen to be rounded up above the sheet or flattened on its surface. I n the thicker sheets, the free cells could be found between the layers of fibroblasts. The association of the free cells with the flattened fibroblastic cells could be observed even in the smallest clumps (figs. 54, %, 55-67,

The fibroblasts that formed the actual cohesive sheets were of two morphologic types; one formed an epitheloid sheet (fig. 16), the other a typical connective tissue sheet (fig. 17), and when the two cell sheets grew in close apposition to each

73-79).


other, a pattern was formed that was very similar to the edge of a growing tumor (fig. 41). The difference in the size and character of the two cell types is illustrated in figure 42.

The cells surrounding the organized clumps were classed as macrophages from their morphological appearance, and like macrophages they were very pleomorphic (figs. 20, 23, 34, 36, 37, 49, 51, 52, 53, 67, 68). They were round or long, big or little, and could have large or small, single or multiple nuclei. There were areas where many small macrophages were clustered together and there were areas where they were widely separated (fig. 15). Of greatest interest, however, was the fact that after 5 to 10 days, virtually the only cell seen separately and not associated with the cell sheets was the macrophage. All of the other free cells were associated with the cell clumps.

A sudden burst of mitotic activity was associated with the formation of the cell clumps. Prior to this time, during the first three days, mitoses were rarely seen, but once the cells began to organize, mitoses were abundant and continued to be so for about the first three weeks (figs. 39, 44). I n older chambers, mitoses were reduced in number and they ulti- mately appeared to reach a constant low level. The thick cell sheets seen at 140 days were made up of healthy cells; there was virtually no necrosis and no cell debris was seen.

Cell modulation. After 24 hours in the chambers when the cells were examined with high microscopic magnification, only the original 5 types were recognized (figs. 18-22), but during the next several days, transformation in morphology and cell behavior occurred which led to the formation of the mature growth pattern just described.

The free macrophages introduced into the chambers rap- idly acquired a very active plasma membrane and a PAS- positive cytoplasm filled with numerous small vacuoles (fig. 20). These vacuoles contained lipid and plasmalogen or, frequently, ingested cell debris. The nuclei were usually small in relation to the cytoplasmic volume, round or oval in shape, and contained several dark nucleoli in dense, finely dispersed


chromatin. Modulation of these cells into fibroblasts occurred in single isolated cells or in groups of cells (figs. 34, 37) ; in so doing, the nuclei enlarged and one or two prominent nucleoli appeared in a pale nucleoplasm. The cytoplasm lost its frothy appearance, and although many of the large vacuoles containing ingested material still remained, the cytoplasm became smooth and basophilic (figds. 42, 44-47). While these cells usually become part of a sheet of fibroblasts, an occasional lone hirtellous cell was observed (fig. 48).

The changes occurring in the lymphocytes were more complex and difficult to follow, but analysis of the morphology of the cells in chambers of different ages provided evidence that the lymphocyte could transform into a macrophage, into a fibroblast via the macrophage-like cell, o r into a plasma cell either directly or indirectly. I n the transformation of the lymphocyte into a macrophage or fibroblast, the cytoplasm increased in volume and tended to spread out and become vacuolated (figs. 23-28, 31-33, 35). Cell processes extended from the edge of the plasma membrane and tenuous contact was frequently established between the cells. When trapped in the meshes of the fibrin fibers that were present in the chambers, the cells elongated and stretched out thick cytoplasmic processes (figs. 33 and 57). Cells of the latter type were still seen at 140 days (fig. 55).

After 4 or 5 days the enlarged lymphocytes were deeply stained by the P A S reaction and the many free floating cells in the chambers appeared as dense scarlet spheres. The cells that were spread out on the filter surface were so deeply stained that the nucleus was frequently obscured. Compare figures 23 and 24 and note that the size of the macrophages remains constant while in the two-day interval represented by the photographs, the size of the lymphocytes has increased. The small dense cells in figure 24 could no longer be identified as lymphocytes on the basis of their morphology, but comparison with the cells in figure 23 leaves little doubt that they were originally lymphocytes. These cells were a little larger than lymphocytes, they had a PAS-positive cytoplasm


that usually was vacuolated but also could be smooth and free of vacuoles (fig, 24). The cells were frequently seen to have an undulating membrane. Free floating cells of this type formed sheets by establishing contact with other cells and becoming sessile. Figures 38 and 39 illustrate an early stage in the formation of a cell sheet in a P-lymph chamber and figure 40 shows, in greater detail, the same process occurring in a Normal Cell chamber. In the latter figure, the cells are forming a second layer over an already existing sheet of fibroblasts. Through cytoplasmic expansion and cell division, the cells eventually formed solid sheets of fibroblasts which in two weeks ’ time became several millimeters in diameter and several layers thick. The actual extent of the three-week growth of P-lymphs in a rat chamber can be seen in the natural-size photograph of the center filter shown in figure 14.

Concomitant with the formation of fibroblastic sheets either by the macrophages o r by the lymphocytes, plasma cells appeared. They were first seen at three days and were present at all time intervals thereafter. After the cells were put into the chambers, most of the lymphocytes were free-floating and they could be found in a “puddle” on one side of chambers opened during the first 72 hours. When the cells began t o organize into cell sheets and the viscous extracellular matrix was produced (see later), large aggregations of plasma cells were found where formerly lymphocytes could be seen (fig. 54). Thus the evidence indicated that at the early times, at least, the plasma cells arose from lymphocytes by the process of modulation. It is worthy of note that with rare ex- ception plasma cells occurred only in association with the cell clumps. Above the cell clumps, plasma cells of various sizes and shapes were seen; some contained Russell bodies, many shed blebs of cytoplasm, and on extremely rare occa- sions they were seen in mitosis (figs. 33, 50,55, 56, 59, 60,79).

The fate of the mast cells and granulocytes (PMN) in the chambers could not be followed with certainty, mainly because they represented such a small proportion of the cells


introduced into the chambers. Mast cells were found in many but not all chambers up to 4% months (fig. 50), and although PMN were not found at this time, granulocytopoiesis was observed at earlier times. I n the P-lymph chambers, espe- cially, PMN were found in clusters and cell division was frequently observed (figs. 29 and 30). I n the two-week rat chamber containing mouse cells, granulocytopoiesis was particularly prominent. Growth was extensive in this chamber and numerous large patches of PMN with typical doughnut- shaped nuclei were sprinkled in clusters on top of the fibroblastic sheets (figs. 69-72). It could not be determined whether these foci arose from PMN that were originally put into the chambers or by modulation of large cells that appeared to be emerging from the fibroblastic layer. With regard to the latter possibility it could be seen that near the edges of the patches of PMN, partially rounded-up cells, apparently coming from the underlying cell sheet, were particularly abundant (fig. 72 at top; fig. 73). Also located in the same vicinity were large basophilic cells in whose nuclei small holes could be seen (fig. 70). Mitotic activity was extraordinarily high in these foci of PAIN and there was a wide range of cell size.

I n the 14-day-old rat chamber, a variety of other free cell types were observed (figs. 7478). Some of these were readily identified as lymphocytes ; others might represent stages in the lymphocyte to plasma cell transformation. Figure 79 shows two plasma cells in very late telophase of division. Compare the cells in figures 75, 77, and 79 with figures 7, 8, and 10 of the recent paper by Nossal ( '59). Nossal's description of the stages in plasma cell formation in rat lymph nodes could be applied with equal accuracy to the cells shown here.

In older chambers, lymphocytes were very scarce and were usually seen as isolated cells in the fibrous matrix of the cell clump (figs. 61-64). The predominent free cell in the old chambers was one that resembled a macrophagc when spread out on the filter, but near cell clumps, these same cells could be traced through various stages of modulation into plasma


cells (figs. 65, 66, 68). Thus in old chambers, the evidence pointed to the macrophage-like cell as the precursor of the plasma cell.

Control chambers used f o r microscopic study of the attachment of the peritoneal cells of the host to the outside of the chambers were left in the mice for no longer than 10 days because after this time the cell layers became too thick to study in the ‘ ‘whole mount’ preparations.

Twenty-four hours after the control chambers were placed in the animal, the outer surfaces of the filters were covered with an evenly spaced, single layer of cells made up largely of neutrophilic and eosinophilic granulocytes (fig. 5 ) , with about one-fourth as many macrophages and a sprinkling of mast cells. Lymphocytes were found at this time only on those chambers that were attached to the liver or mesenteries. They were flattened on the filter or rounded up and slightly above the filter surface (figs. 6, 7). As time progressed sinall groups of fibroblasts formed closely-knit groups (fig. 8) among the dispersed cells. These groups increased in size and thickness and in due course covered the whole chamber with a connective tissue capsule. The discrete cells - granulocytes (fig. l o ) , mast cells, lymphocytes - were seen in association with these islands of connective tissue and it was here that plasma cells were first observed (fig. 9). I n the attached chambers, plasma cells were first observed as early as three days, while on the free chambers they were not seen until after 6 days.

Failure of cells f r o m peritoizeal cavity t o pernetrate very dense filters. Thirteen of the 14 control chambers removed after 145 days in the mice were intact and no trace of cells could be detected on the center filters. I n the 14th chamber, a macroscopic hole punctured in the outer filter had permitted the entry of peritoneal cells. The cells that were growing luxuriantly on the center filter were entirely fibroblastic.

Chamber fluid a i d cell secretion. When the top filters were cut away from chambers in which cells had grown for

At tachment of cells t o outside of chambers.

STUDY IN CELL MODULATIOR 295

several days or longer periods, extremely riscous fluid was found therein. The viscous material produced by the cells growing upon the center HA filter, penetrated it, and accumulated on the empty side of the chamber. The fluid nearest tlie peritoneal cavity was viscous and could be pulled into strings of over a foot in lcngth, while against the center filter lay a layer of jelly so firm that it could be picked up with forceps. This material was PA8 positive, even after prolonged soaking in alcohol, and after dialysis against distilled water, gave a typical protein absorption curve. Al- though viscous fluid was not found in control chambcrs en- cIosrd with Very Derrse filters, macro- and microscopically visible fibers were. These fibcrs were probahly cornposed of fibrin similar to the fibers described by Amos and Wake- field ( ’58).

Tlie viscous material was probably R glpco-protein polynicr of such dimensions that it could penetrate the HA filter Init not the Very DevhvLse filters. This was deduced froni tlie fact that tlie fluid in tlie control double chiimbcrs was the coil- sistcricy of blood serum. Since these ch inbers were encap- sulated by a thick fibroblastic sheet, it is cvidciit that any viscous material produced by cells growirg on the out&k of the chamlm-s had not pcrietratd the T’cr!y 11emc: filters.

I n “youiig” chambers, there was evidence that the macrophage might be responsiblc for the production of thc! viscaous niatc>i.id. In the P-lymph charribcrs clunips of mac~*opht~ges \YPYC surrounded by a haze of amorphous PAS-positive material and firic mcthyl-blue-stainccl fibers (figs. 13, 30, 36) and individual macrophages appeared to leavc behind n, trail of tliis secretion. Whether these cells continued to produce tliis material could riot be ascertained but it was interesting to observe in older chambers another functional capacity of the macrophage, namely the capacity to digest thr thin layer of fibrin that covered the center filter. Individual cells n-ere surrounded by a clear zone and worm-like tracks vere le f t 11y moving cells (figs. 49, 51, 53).


Hydroxyprol ine analysis. No hydroxyproline (OHP) could be detected in extracts of normal peritoneal cells. Two separate analyses on extracts of 28 and 31 million cells, respectively, showed no OHP spot on the chromatograms. Since about one-tenth of this number of cells was put into the chambers, it is certain that no detectable ORP was introduced with the cells. In addition, since hydroxyproline was not detected in empty chambers that had been in the animal as long as 131 days, it seems safe to coriclude that the OHP that appeared after the cells grew for two weeks was the result of new synthesis of this amino acid (table 4). The range of the OHP values obtained was large but was in ac- cord with the great variation in the size of the fibroblastic sheets. On the average, there was a consistent increase in the amount of OHP in the chambers with the passage of time.

TABLE 4

duerage hydroxyproline contcnt of normul cell cliumbers

TIXE IN NUMBER O F AYKRACTC OHP A N I M A L CHAYRTCRS PER CHAMBER’

dnys P!l

15 4 7.5 (5.0-8.9) 28 6 15.0 (6.423.7) 43 5 22.6 (6.145.3)

. ~~

Figures in parentheses give the range of the analyses.

DISCUSSION

I n 1888 Elias Metchnikoff published his observations on experimental tuberculous lesions and described the hyper- trophy of lymphocytes into phagocytic macrophages. Maxi- mow ( ’02) not only confirmed Xetchnikoff’s observations but also described the transformation of the lymphocyte into a multipotent cell, the “polyblast” which in turn had the capacity to become a fibroblast. This thesis met with such vigorous opposition that Riaximow ( ’28) in the introduction to a more definitive paper felt compelled to write as follows: “ I n my first paper on inflammation, in 1902, 1 emphasized, that the lymphocytes as well as the monoeytes can transform

STUDY I N CELL MOIlULATION 297

theniselves into phagocytic amebocytes, tlie so-called poly- blasts, and further into fibroblasts or ~omiiio~i fixed connective tissue cells. I t is very doubtful whether these new experimental facts will convert the “diehard ” representdatives of orthodox hematology . . .,, This statement has equal per- tinence today, because in spite of the many experiments performed in the 31 intervening years (see reviews of Ko- louch, ’39; Rebuck, ’47; and Trowell, ’ 5 8 ) , tlie definitive ex- periment of showing the transformation of an individual lymphocyte into a fibroblast has yet to be performed.

The diffusion chamber technique, however, has provided new evidence in support of the lymphocyte-to-fibroblast transformation. The technique has permitted us (a ) to grow cells of known nurriher and type for long periods of time in an isologous or Iicterologous environment, (b) to cxamine cell relationships in stained, whole-mount preparations, and (c) to analyze chemically the contents of the chambers.

In his recent review, Trowell (’58) states that “there is no real cvidencc that lympliocytcs can tur.11 into fibroblasts, even via monocytes.” It is our belief that we have presented in this paper, pictorial and chemical evidence that this is possible. I n considering the evidence in this direction, one point should be emphasized, namely, that while Maxirriow ’8 thesis has not been accepted even at thc present time, Metch- nikoff’s has. Thus Trowell, after citing the pertinent recent work, states “. . . there is fairly good evidence that lymphocytes can turn into monocytes and macrophages . . .,’ I n the diffusion chamber the lymphocyte modulates into a cell with a vacuolated, PAS-positive cytoplasm, the cell that Maximow called the polyblast. The polyblast has been observed by many workers under a variety of conditions and there is no question that it is a real entity-no matter what name is applied to it. I n the diffusion chamber, it is this cell, modn- lated from a lymphocyte, that has the capacity to go from a state of free floating independence to the condition of sessile, communal existence of a sheet of fibroblasts (figs. 38-40). The phenomenon is most clearly observed in chambers where

298 EMMA SHIELTON AND MARY E. RICE

P-lymphs were grown and where the modulation of the lymphocytes was not confused by the modulation of other cell typcs. The electron micrographs of the fibers produced by the cells and the hydroxyprolinc analysis of the tissue that grew in the chambers lcave no room for dispute that collagen producing cells, fibroblasts, were present.

Not all of the fibroblasts in the chambers came from the lyriipliocyte via the polyblast, rriariy modulated directly from the inacrophage, and it is intcrcsting to speculate upon the physiological significance of the niorpliologic laterations that were scon in the lyrnphocytc a id niacrophage during the process of modulation. Evidence of direct ti~ansforniation of the lymphocyte into a fibroblast wns not observed, because before the lynipliocyte became IL fibroblast, it acquired a vacuolated I'iWpositive cytoplasm arid could no longer be called a lym- plivcytc 011 the basis of its morphology. The macrophages already had a PAS-positive vacuolated cytoplasm. In the actual transformation into a collagen producing cell, thc I'AS-I)ositivc vacuolntctl cytoglasrn was lost by both of these cells, ant1 replaced by a sniooth, basopliilio cytoplasm. This suggested that the csseritial physiology of the transformation involved tlir cessation of piiiocytosis and the concornitant stor- age of materials in the form of glyco-protein, and the con- version of tlie cells ' metabolic energies toward protein spn- thesis, the formation of collagen.

I f the fibroblasts in the chambers did not come from the lymphocytes and macrophages through the process of rnodu- lation, where did they come from? The possilnility must be consiclrrecl that the collagen found in tlie chambrrs was produced by cells that originated from one or several fibroblasts that were by accident included in the frce cclls of the peritoneal fluid. TTVO considcrations appear to make this possibility a remote one. The first is a statistical consideration and the second conccrns the manner in which the cells grew in the chambers. From the statistical point of view, con- sistently large numbers of fibroblasts would have had to be present in the mouse peritoneal fluid in order to contarninate


each of the 126 chambers examined in these experimeiits- aiid this despitc the fact that no fibroblasts were observed during dif'ferential counts involving 27,000 normal peritoneal cells (table 1).

Perhaps of more importance was the niorpliologic appearance of tlie cells at different time intervals. The general im- pression obtained from studying the stained filters was that while all of thc cell modulations occurred very soon after the cells were put into the chambers -- certainly during the first two weeks-cell modulation did not thcii stop. Tn many of tlie chambers opened after 4Yz months, very small islands of fibroblasts were still being formed by a process that was in every way comparable with that secn after the cells Iiad been in the chambers for only a few days. It appeared then that the fibroblasts could arise from the modulation of the lymphocytes and macrophages and further, that thc modulation could occur at ariy point in time, triggered by some unknown mechanism.

Another typc of cell modulation observed in the chambers was the formation of plasma cells. There lias been coiisjder- able speculation in recent times as to tlic exact origin of tlie plasiiia cell (Fagraeus, '48 ; Kucning and vaii der Sliklre, '30 ; arid Trowell, '58), that is, wliethcr it comes from the transformation of the lymphocyte o r from a reticular cell 01' mat- rophage. The events occurring in the chambers -- the early appearance of plasma cells arid thcir relationship in space to areas where the lymphocytes congregate -led us to be- lieve that the lymphocyte could transform dircctly into the plasma cell. I n the older chambers thcre was equally con- viiicirig evidence that they were arising from the macropliage- like " polyblast. " Here thc successive stagcs in the formation of the plasma cell from the fixed polyblast were observed by thc mere expedient of travcrsing with the microscopc the areas beside and on top of the various ccll clumps.

Recent work lias implicated the plasma cell as the cell which produces antibody protein ( Fagraeus, '48 ; Keuning arid van der Slikke, '50; Leduc ct al., '55; Nossal, '59). but the

300 EMMA SIIELTON AND MAEY E. RICE

appearance of plasma cells in abundance in the isolog oous c11-

vironmcnt of the mouse chamber raises tlic question of tlie reason for their formation. This is not the first tirile that the question has becn raised. Policard et al., (’54) observed in ra ts a dramatic accumulatioii of plasma cells in response to the ititraperitoiieal injection of poivdercd quartz. These authors also were puzzled by the apparent contradiction prc- sciited by the appearance of large numbers of antibodp-producing cells in response to a nonailtigenic stimulus. Both observations appear to be a demonstration of the response of the free cells of tlie rcticulo-eridothelial system to iiii inert material .- the so-called foreign-body reaction. Whether antibody is formed by plasma cells iii this situation remains to bc demonstrated.

Interaction between cells was an irnportaiit feature of tlic cell modulation seen in the chambers. The initial cell modulations that took place in tlie cliarnber were probably pro- voked by what is termed vaguely “the adjustment to a new cnvironmeiit, ” a plieiiomenoii ~ ~ l l - k n o ~ n in tissue culture and about mliicli very little is understood. Once the adjust- rrient was made, howevei., a new pattern of growth was established aiid evidence of cell interaction became clear. Thus the sheets of fibroblasts that very soon developed in the clianibers were centers of niutual cellular influence. Tt was herc that plasma cells were found, he re that granulocytopoiesis occurred, lierc that lyrnphocytes and mast cells were found in the old charnbcrs. Only iii the clunips \wre cells of different types to be found. Sway from the clumps aiid scattered on the filter w r e macrophage-like cells which seemed in their isolation to hc mutually repulsive, yet in the vicinity of the cell clumps tlicsc same cells would rouiid up, float free, and under tlie influence of thc new environment become plasrria cells.

Evidence for the interdependence of cells is multitudinous. Embryonic diff erentiation is based upon it (Weiss, ’39) and tlie recent work of Moscona arid Illoseona (’52) and Grob- stein (’56) has attempted to elaborate the mechanism of this


interdependence. Evidence has also accumulated in the past few years which indicates that cells of the adult animal are not static end-products of differentiation but may be influenced profoundly by their environment. Leduc ( '59) has demonstrated the modulation of the highly differentiated adult liver cell into the bile duct cell, and Chase ('58) has shown that cells of the external sheath of the hair follicle of the adult mouse have a variety of potentialities that are realized in direct response to variations in tissue environment.

Sliould the free cells of the reticulo-endothelial system be exempt from external influence? It is reasonable to assume, on the contrary, that by virtue of their mobility, they would be the cells most easily influenced by an ever-changing environment and that they would respond to the abnormal environment created by a wound, a tumor, infection, o r even metabolic disturbance in a manner not unlike that observed in the diffusion chamber.

SUMMARY

The diffusion chamber technique has been used to study the growth and modulation of the iiorinal f ree cells of the peritoneal cavity of the Strain A mouse. These cells - lymphocytes, macrophages, granulocytes, mast cells and an occasional transitional cell - maintain a remarkably constant proportion relative to each other. The diffusion chamber is constructed of cellulose membrane filters supported on plastic rings, and when it is placed by laporotomy into the peritoneal cavity of an animal, fluid enters but peritoneal cells are excluded. Thus, cells placed inside the chambers are nourished by the peritoneal fluid of the host without being contaminated by host cells.

When the free cells of the peritoneal fluid of mice are grown in diffusion chambers, transformations occur which lead to the formation of an organized tissue. Evidence is presented which indicates that the lymphocyte modulates into an in- different cell which, in turn, has the capacity to loccome a

302 EMMA SIIELTON AND MARY K. RICE

fibroblast or a plasma cell. The macrophage also appears to have the same capacity. Crranulocytopoiesis occurs in the chambers and while niitosis was not observed in mast cells, they are still present in the chambers after 140 days. Host cells attaching to the ouside of the chambers iindergo similar alterations.

Collagen was identified in the chanibers by clcctron microscopy and chemcial analysis aftcr the cells had grown for two weeks. The collagen content of the chanilws increased with time.

The nature and significance of thcso cell modulations is discussed.

RCKNOW1J':DUMENT

The authors wish to express their appreciation to I. 1,. Bragg, R. R. Padgett arid other members of the Photographic Section, National Institutes of Health, fo r printing the photographs used in this paper.

LITERATURE CITED

AMOS, B. D., A N D J. D. WAKEFIELD 1958 Growth of niouse ascites tumor cells in diffusion chambers. I. Studies of growth rate of cells and the rate of entry of antibody. J. Nat. Cancer Inst., 9 1 : 637-670.

1958 Radiation damage to cells dependent on their tissue environment. Brit. J. Badiol., 31 : 65- 69.

1948 The plasma cellular reaction and i ts relation t o the formation of antibodies t n mfro. J . Immunol., 58: 1-14.

1955 A phase-rontrast microscopc study of free cells iiative to thc peritoneal fluid o f DBA/2 mice. J. Nat. Cancer Inst., 16: 415445.

GOLDIE, H., AND M. D. FELIX 1931 Gioivth charactelistics of free tunior cells transferred serially in the peritoneal fluid of the inuuse. Cancer Res., 17: 73-80.

GROBSTEIN, C. Transfilter induction o f tubules iii mouse metanephric mes- enchyme. Exp. Cell Res., 20: 4'74-440.

HIRSCIIFELD, H. 1927 Ziicbtungsversuche mit freien Eu(1atszelleii. Arch. f. exp. Zrllforsch., 4 : 438-441.

KEUNING, F. J., AND L. B. VAN DER SLIKKE The role of inimature plasnin cells, lymphohlasts, and lymphocytes in the formation of antibodies as established i n tissue culture experiments. J. 1,ab. Clin. Mcd., 36: 167-182.

CHASE, H. B.

FAGRAEUS, A.

FELIX, M. D., AND A. J. DALTON

1956

1930

STUDY I N CELL M0L)ULATION 303

KLEIN, G. 1951 Comparative studies of niouse tumors with respect to their capacity for growth as ‘ I ascites tumors’ ’ aiid their average nucleic acid content per cell. Exp. Cell Rcs., 21: 518-373.

K(JI,OUCH, F. 1939 The lymphocyte in acute inflammation. Am. J. Anat., 15:

LEDUC, E. H. 1959 Cell modulation in liver pathology. J. Histocheui. Cyto- cliem., 7: 253-256.

LEDUC, E. H., A. H. COONS AND J. M. CONWOLLY 1955 Studies on antibotly production. 11. The primary and secondary responses in the popliten1 lymph node of the iabbit. J . Exp. Med., 102: 61-72.

LILLIE, R. I). 1954 Histopathologic Technic and Practical Histochemistry. Blakiston Co., Philadelphia, pp. 356-358.

MAXINOW, A. 1902 Experimentclle Untersuehungen Uber die entziindliche Neubildung von Rindegewcbe. Beitr. z. Path. Anat. u. z. Allg. Path., (Suppl.) 5 : 1-269.

Cultures of blood lencocytcs. From lymphocyte :nld rnonocytt. to connective tissue. Arch. Exp. Zellforsch., 6 : 169-268.

METCHWIKOI F, E. 1888 fiber die phagocytare Rolle cler Tuljerkelricszellen. Virchows’ Arch. f . Path. Anat., 113: 63-94.

kfOSCONA, A., AND H. MOSCONA The dissociation and aggregation of cells from organ rudiments of the early chick emhiyo. J. Anat., 86:

NOSSAL, G. J. V. 1959 Antibody production by single cells. 1x1. The histology of antibody production. Brit. J. E K ~ . Path., 40: 301-311.

POLICARD, A., A. COLI~ET AND L. CILTAIRE-RALYTE 1954 Etude au microscope electronique de l’action dcs poussibes de silicc sur les plasniocytes ct les cellulcs reticulo-histiocytaires dcs mammifkres. Revue d ’Hematol.,

PORTER, K. R., AND G. D. FAPPAS Collagen formation by fibroblasts of

IIEBUCK, J . W. 1947 The functions of the whi t r hlood rrlls. Am. J. C h i . Path.,

ROBERTS, 11. R., M. G. ROLOR AND W. B u s ~ r c 1958 A rapid paper chromato- graphic procedure for the quantitative determination of hydroxyproline. Nature, 182: 1602.

1958 Studies on moiisr lymphomas. 11. Behavior of three lyniphomas in diffusion chambers in relation to their invasivc capacity in the host. J. Nat. Cancer Inst., 27: 137-161.

TROWELL, 0. A . 1958 The lymphocyte. Internat. Rev. Cytol., 7 : 236- 286. \VEISS, P. 1939 Principles of Development. Henry ITolt and Company, Kern

41 3-428.

1928

1952

28 7-30 1.

9 : 403-417. 1959

the chick embryo dermis. J. Biophys. Kiochem. Cptol., 5 : 153-166.

1 7 : 614-630.

sJI%ELTON, E., AND M. E. lZIcE

York.

PLATE 1

EXPLANATION OF FIGURllS

Phase contrast photographs of the free cells of the pcritoiieal fluid of the Strain A Mouse. X 2000.

1

2 Neutrophil graiiulocytes (in motion) slid lyinphocyte : inarcrophages ( M )

Lymphocytes and inacrophage with expanded cytoplasm.

and a transitional cell ( T ) f r o m pcritoncal cavity containing a chamber.

3 Eosinophil granulocyte.

4 Mast cell.

304

STUDY I N CELL M O D U I A T I O K E M M A SHELTON A N D MARY E . RICE

PLATE I

305

PLSTE 2

EXPLANATION OF FIGURES

Control chambers. Cells 011 outer surface of filter.

5 Graiiulocytrs and histiocytes a re dispersed evenly on thc filter surface. Com- pare with figure 11. 24 hoiirs. X 150.

Near a point of attachment of the cliamber to the liver, neuiiierous lymphocytes accumulate. 24 hours. X 150.

Lymphocytes make coiitact nitli each other through tenuous cytoplasmic pro- cesseb. Conipare with figures 20, 22, 23. 24 hours. X 2000.

“ R a f t ” of cells in wliieli can he recognized lyinphocytes and fibroblasts. 10 clays. X 800.

6

7

8

9 Plasma cells and unidentified cell. 10 days. x 1000.

10 Patell of polyniorphonuelcar leucocytes. 10 days. x 800.

S T U D Y IK CELL M O U U L A T I O T E X M 4 SHbI'TOX A N D DI 4111 b. R I C E

PLATE 3

EXPLANATION OF F I G U R E S

11 Normal Cells after 20 hours in the chamber. The flattened cells arc micro- phages and granulocytes. The dark dots are rounded-up lymphocytes. ?< 130.

12 P-lymphs a f te r 46 hours in the chamber. Observe that lymphocytes are predominant cell. Compare with figure 11. x 150.

P-lyinphs a f te r three days iii the chamber. The rnacroplisges are prominent because of the PAS-positive material surrouridiug them. x 150.

13

308

STUDY I N CELL MODULATION E M M A SHELTON AND MARY E. RICE

PLATE 3

309

PLATE 4

E S P L l X A T I O X OF FIGURES

' ' Matn1c pattc111 of pi O \ \ i h . ' '

14 Actual dimensions of the center filter of a ra t chaniber slioiiiiig the extciit of growth of iriouse P lgniplis after tlircc neeks in the animal. Initial inoculuni 2.3 X 10" cells; natural siie.

Appeaiaiice of Norinal Cells after tliicc weeks in inouse chamber. The cells ale glowing i n clumps that a l e surioiuidecl by dispeiscd. single cclls. X 100.

Edge of large cell sheet n l ic ie cells gioviiig j i i an epitlielioicl pattern. P- lyiiiplis in rat chamber. 7 (la) s. x 100.

Etlge of cell slicet i l l nliicli cvlls n c i e gro\\iiig in tjliicullg fibioblastic pattern. Kornial cclls. 8 days. x 100.

13

1 6

1 7

310

STUDY I N CELL MODULATIOK B N X A SHELTON A N D M A R S E. RICE

PLATE 4

311

Detail of crlls aftclr 24 Iiours in clialiibers. X 20(10.

18 Lyriiplioeytes of various sizes.

19 Nas t crll, with granules :ippc:iriiig as iicgntivc inlnges, surromiilcd by lpi- plioeytcs aiid a macrophage. N o t e tlic iiiitiniate coiitact cstablislicd between tlic surrouiicliiig eclls an(l the Iii;!st cell. T h i s react ion to ~ i l a s t crlls W:LS

:I reyul:irly observed plieiionierion.

PLATE 5

313

STUDY IX CELL MODULATION EDlAIA SHELTON AiTD Y A l & Y E. RICE

PLATE 6

EXPLANATION OF FIGURES

20 Macrophagcs, lymphocytes, and a transitional ecll (’I’) after i U hours in the chamber. X 2000.

2 1 Lymphocytes aiid gmnulocytes. 24 hours. Y 2000.

22 Lympliocyfcs 011 rl, background of fibels. 24 hours. X o t e mitotic figure. x 2000.

314

STUDY I N CELL I\IODULA'l'lOII E Y X A SHbLTOX A l l U J141ZY E. R I C E

PLATE 6

315

2.3 Lpiphocytrs niitl uiucroyhngca nftcr thrrc tlatyn i i i chaiuber. S o t e tho teuuous contnct estnbliuhed bctwrcii iiiniiy of the l;miplioc;rtc~s. A fcw iiiaut edls nitli uiistsiitrtl granules c:iu $1180 bc uccu. X 500.

Tlie rcl:it.ioiisliip beti~cmi the siiitill, doiso cells in this piet.urc and the 1 y - pliocytes in figure 23 is uniuhtnknblc. The magni8catiou of both figures is the s:iitic niid the she of thc macrophages in each photograph is the same, but the Iyinphoeytes havo iiicrcnsed ju size niid acquired n PAS-positive vticuol:it.cd eytupllasm; eoiiipnre with eelL in figure 39. The smtill &-we cells iii this IJhotOgrph arc inorphologieiilly iiot 1.vmphOcytes but macrophages (or the ‘ ‘ polyblast ’ ’ of Masiiiiom, ’02). The suiall rouud cells (arrow) that are flontiug above the fliittciicd cells tire ickiiticul with those sho\vu by Hirwh- fdtl (’27). 5 clays. X 800.

25, 26, 27 aiicl 28 P-lyinplis after three days iii clinnibers. In figure 25 the cyto- plnsiu of a siii:t11 macrophage appears to be surrouding a lymphocyte. la figure 27 coitiparc the clizc of the large macrophage out of focus in lower right corner, with that of tho 1ymplioc.ytes in the process of modulating into small macrophagos. Bigureli 26 aiid 28 show cytoplasmic expusion in two S l l d l Ipphocytrs. X 1000, X 800, X 800, 2( 2000, respectively.

24

316

31 7


PLATE 8

EXPLANATION 01) FIGUZES

P-lyniplis after tliree days in the ehainber. X 1Od0.

29 n ~ i d 30 Groups of p01yniorphonucle:rl. lcucocytcs : n r d lyitiphwytes. T t was iii

clustcrs such 11s these tliiit mitotic activity was espcci:illy noticeable. The haze nrouiid the 1ii:icroplinge in figuie 30 is €'AS-l,ositivr.

31 and 32 111 these clusters of IymIhocytcs all stngcBs of cytoplasniie eq~:uisiioii c:ui he observed.

318


PLATE 8

319

PLATE 9

EXPLANATION OF FIOmES

33 These cclls are sprcucl out jn a fibrous gel. The fibers arc probably fibrin. Many of these colls can bc rccogniml as modulating lymphocytes. Two plasinn cells in the proceas of shedding cytoplasnlie droplets arc to be seen slightly left of ccntor. Coiupa.rc with figure 5i . Nomial Cells at three clays. x 1000.

34 In the muno c.limilwr as Hhowii in figure 33, iiiri.cropliages spread out on the filter surface. At thc upper right are two cells that were probably orig i d l y lyrupliocytex. In the center are two macrophages niodulating into fibroblnsts. x 1000.

35 Macrophages, lymphocytes, and modulating lymphocytes. Observe the e;).to- plamic processes on the two cclls at left below ccutcr. P-lymphs a t thrce days. x 800.

320

321


36 Chimp of ii1:tcorpIi:igrs iii P-lyiiiph climil)er. There is 110 evidcnce of orgxiiim- tioii i n t,liis clump of cells, nor do tlic lyii~p110 :~ppcar t o be n t t m d c d to it. Note the PAS-posit micling the inac~ophages. Tlirce days. X 800.

Tii coirt,rast t o t h e ii1:~cropli:igcs ~ l i o x i i in iigurc 3f;, t!ic cells iii this cluiiip shun cldmite orgmkmtioii miti oriciitatiun. This repwsciits the niociulation of niacrop1iagc.s into fibrobkists :ind the early stngr of sliert forination.

of tllP chllllp. Ntnnlal Cells at

37

e fouiitl only iir tlicl viciii

STUDY I N CELL XODULSTION E X M A SHE l iTOX A A D M A R Y E R I C E

PIATE 10

323

l\lotlulation of lyiripliocytcs iiito fihro1Jl:ists.

38 P-lyiiplis a f te r 8 clays in the c1i:Inlljer are brgillllillg to form 3 solid ijlleet of crlls. x 100.

'1 of cclls :it edge of cell sheet s l io \~n i n fignrc 38. ('oiiipare nioypliology of tlicsc. cells with the sm:rll dense cells i i i figures 24.

E'rec-floating crlls attach tlienisclves to cells ;it tlir etlge of the cell slieet. Thew cells nl'e forming ;tilotlirr laycr a h v e :in alreatly existing sheet, of cells. Coiiipnrc tlic size aiid 111or~~l~ologg of thew crlls \Tit11 that of the Iymphorytc:? nftcr 24 hours ill the cli:inibcr, figures 1s-22. rl'li(, size of the wlls lins onlg sliglitly iiicrensecl but the morp1iologir:il Li1ter:iti:iii is obvious. Normxl Cells, 8 tlnys. >< 2000.

x 800.

40

304

325


PLATE 12

EXPL4NBTION OF FIGUBES

41 Junction between epitheloid and fibro1)lastic cell sheets. P-lymphs in rat, 7 days. X 100.

42 Detail of cells at junction between cell sheets shown in figure 41. Note differelice in cell size. P-lymphs in rat, 7 days. x 500.

Electron nlierograph of collagen fibers between two cells. The period in the fibers i n roughly 423 A ( w e Porter and Pappxs, '69, p. 161). P-lymphs in mouse, 18 days. About X 43,000.

Mitoses are frequently observed among the cells a t thc edge of the cell sheet. Observe cell inclusions. P-lymphs in rat, 7 days. x 500.

43

44

327


PLATE 13

EXPLANATION O F FIGURES

f’critoiieal 1yiiiphoc.jtc~s iii tlic i a t f o r 7 days. X 800

45 Cells at the very edge of a cell hheet. The two cells in tlic center are typical imicrophages with foamy CytopLibiii xiid mmy small nucleoli in the deiisc nuclei. To thc right sic' three c~’lls that reprrsent stages of trailsition to fibroblasts; theii nuclci ale palrr :iiiil contain inore p~oin jnent nnelcoli, nhilc their cytoplasm is less vncuolatrd.

-16 A t the cdgc of cell sliccts, cclls like these are frequcntly obscrved. The plasma. iriciiil~raric is d r a r c i i into n multitude of hair-like eltensions and the edges of tlic cell are so tliiii aiicl tcnuous as t o be almost uncliscernible. Aroiintl the cell nncleus arc inmiy vaciiolrs containing the remains of pliagocyti7ed cells. Tlic i i i i c k ~ i ~ itsvlf is a typieal fibroblast niicleus.

47 In tlic center of n singlc lnycrcd crll sheet, tlir cells acquire rz pattern reniinisccwt of squmious cpitlieham. Observe the relationship between the “ f r w ” cells aiid the eells niakiiig up the cell sheet. The space between the wlls ii probaljly causcd by the stretching of the filter during the htaining procedure.

338

S T U D Y I S CELL IIIODULATTON EM ~ r . 4 sIII:moN ASD n l a a ~ L. RICE

PLATE 13

329

PLATE 14

EXPLAXATION OF FIGURES

48 A single, large cell with the plasma nienibrane drawn into iiuiiicious filament- ous ewtensions. Cells of this type were observcd with moderate frequency. P lymphs in rat, 14 Clays. X 500.

49 Several fibroblastic cells rail be coiitrastcd I i r w with two rnacropliages. P-lymphs in rat, 14 days. X 350.

50 Mast rells are still present in cliambers a f te r 53 days. Notc plasma cells. Normal cells. x 1000.

51 a i d 53 Macrophages leave clear tracks in the protciii film on the filter. P- lymphs in niice, 14 days. X 100 and X 350.

Isolated macrophages arc always present in the chambers. Normal cells after 53 days. X 1000.

52

330

STUDY IN CELL MODVLATIOiX FihlMA SHELTON A X D MARY E. R I C E

PLATE 14

331

B X P L l S Vl’IOK OF F I G U R E S

54 Plasinn cells in large ~iimibcrs arc found n h o w a sheet of fibroblasts. P- lymplis, 10 days. x 500.

h h t u r e plasm:i cells locnted :rbore :L slicct of fibroblasts, the riuclei of which can be seen out-of-focus below. Two of thc cells :Lie fillcd with PAS positiye Russcll bodics. P-lyiiiplls, 14 days. x 1000.

P1:tsma cells are still observed i11 ch:~nilms af te r 110 days, (compare with fig. 54). These cells wcrc :rho associatctl with a slirct of filirohlasts. The fibrous nitturc of the oltlcr cclll sliects is SCCII here. Normal cells, 140 (lays. x 800.

55

56

332

STUDY I N CELL I\lODULATIO?r’ E M X A SHELTON AXD M A R Y 3:. RICE

PLATE 15

333

57 After only thrce days in the cliiiiiibcr iiianp crlls tif th i s type arc serit spread out iii the iiicshes of t l ic fihriii filwrs. Coiiiparc these cells with those iii

figuro 33. The cvitliwce (sizc of the cctllx, tlieir nurnbcr, their location in thc chmnbcr) stroiigly suggrsts that thc cells wcrc originally lymphocytes and that they have gaiitctl their niorphologic apIiearaiice through modulation. Norrn:d cclls. X 1000.

After 140 days cells of this type (ctimpnre wit11 figure 57) are still seeu in the chambers. Norinn1 cells. X 1000.

Maturc plasnia crlls alipear i n grtxt iiniiilwrs ~ v l i v i t prritoiirnl lyinphocytcs arc put i i t the chamhers. P-lyiiiphs, 14 krys. X 1000.

After 140 days, umiiy p1;isin:i rrlls a rc still preseiit. Note tlie g r rn t vnrint.ioii iu ccll sizc. Sornial cells, 1-10 days. X 1000.

58

59

60

61, 62, 83 ;md 61 After 140 tl:lps, lpinpliocytes caii still be fouiitl in tlic fibrous grl above tlic slicets of fibroblasts. Norm:il cells, 140 d:tys. X 1000.

334

335


PLATE li

EXPL-4NATION 01’ FIGURES

Notniai cellz iLftcr 140 d:iys in the sliariiber. These Iiliotographs illustrate the teridency for “ f ree” cclls t o accuniulnte ill tlic aiciiiity of fibroblastic sheets cvcn after 414 months in the altificinl ciivironnieiit of llir chamber.

65 At tlic edge of the shcct of fibroblasts (seen as an out-cf-focus haze oil tho 1c.ft) thc “frc,c” cc,lls Iwgiii to flatten out. Tliese cells arc precursors of pIasma cclls. X 600.

Above a r r r y sriiall siiiglc laycretl diet4 of fihrohlasts iiimiy frce cclls aceti- mulate. Tlic f r w cc~lls arc dcrivcil f m i i t the rlisewti~ wlls that arc spread out on the filtcr arouiicl t he cell shcct. x 500.

Here ;igaiii is ail illustratiou of ccll :tccuniulatiori above a sheet of fibroblasts. Most of the dark dots on thc cell sheet are plasnia cells. x 100.

A t the cdgc of x sheet of Abrol)l:ists, cells that w c w oiice spread out on the filter, round up and float frec.

(Xi

67

68 x 800.

336

337


PLATE 18

ESPLAKATION O F F I G U R E S

These are photographs of iiioube peritoii~al cells g r o v i ~ in a i:it chamber for two weeks. Large sheets of fibroblasts, equal in size t u tliose iliowii iii figuie 14, n c r e foi incd 111 this chamber. Prcseiit above these sheets were nuinerous large foci of di\ idiiig polyinorphonuclear leucocytes ( P M S ) .

69

70

L o w power vicw of a diiiiip of PMIN above the fibioblastic sheet.

Kuuicrous cells of this type were obseivctl near the clumps of PMR. Ob- serve the sinall hole in the ccntcr of the iiuclei of tlicse cclls. It ~voulcl appear that tlicse cells arc the precursors of the PMN.

Cells in tlie ceiitcr of :L group of PRfiY. Note the frequent mitoses a d the variation 111 ccll size. x 1000.

At the very edge of the clusters of PMN, large cvlls appear to he sep,irating themselves from tlie undeilying cell sheet. Two such cells are seen a t the upper right corner. One of them is dividing. x 1000.

X 100.

x 1000.

71

72

338

STUDY IN CELL &IOUULATION EI\IAlA SHELTUN AND DIARY E. R I C E

PLATE 18

Moiise pwitoiirnl 1~iiipliot:ytc~ iii tlir mt. These photograph jllusl rate the raricty of the ccll types obserretl : I~JOVV the shcct of fibroblasts.

73 Tlicse : ire cells located adjaceiit to :I cluster of PJlri. O W is corifroiitcd with tlie question - are tiicy roiulcliiig u p uud sepr:itiiig f rom t,lw re11 sheet, or :ire they settling d o w l :mil becoiiiiiig a part cf it ? In this case the former is probably correct bccausc tlicsc cells npear to IJ~ coiitributing t o the clastein of I’MN. They arc rarely fouiid clscwlicre in the fibro1il;lst sllc-ct. 14 (lays. x 1000. 9 clustw of lyinplioc,ytcs is seer1 on t,lic Icft, while 011 t l r ~ right arc two cells tlmt iiiay be ~ L ’ W L L L ’ S O ~ S of 1’1\7hT.

$4 14 days. X 1000.

-- 1 a Tlicse cells iii:ry br. the precursors of plasiiia cells. 3 1 days. >< 1000.

i G , 7 7 , i 8 and 79 Of the frce cells s l l o ~ l ~ in these pliotogrzrphs only two types c:iii be identificd, the lyinpliocytes ill figure 7G :mtl the plasma rc~lls in figure i9. Tlic otlicr cells illustrntccl hew m:iV bc interiiiedintc st.agcs in the rnoilu- latioii of the lyiiipliocgte t o tlie pl:isnia cell. Note t,hat tlie plasina cells iii

figure 79 :+re i n the late teleplrnsc of mitosis ar!d :ire joined b? LI thin cytoplasmic thread. %’iyures 76-78, 14 (lays. x 1000. Figure 79, 7 tl;iys. X 800.

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STUDY IN CELL 3IODCLITTON hl1nl.k SHhliTOX A i i D M A R 1 E. KICK

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Growth of normal peritoneal cells in diffusion chambers: A study in cell modulation

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