Structure and function phagocytic cells

STRUCTURE AND FUNCTION IN PHAGOCYTIC CELLS

Thesis submitted to

The Australian National University for the degree of

Doctor of Philosophy

by

Robert John North

The Department of Experimental Pathology John Curtin School of Medical Research

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"effected" to read 11_affected" Line 21.

"reprocucability" to read nreproducibility" Line 23

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"transfered" to read utransferredu. Page 103, Line 3.

"transfered" to read "transferred11

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"aoeba" to read uamoeba".

Figure Legends to Paper 7, Figure 3, Line 1.

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The work presented in this thesis was carried out by the candidate.

TABLE OF CONTENTS

INTRODUCTION

_{• • • • • • • • • • • • • • • • • • • •}

SECTION A:

SOME

EXPERIMENTS

CONCERNED

WITH THE

MECHANISM

OF

PHAGOCYTOSIS

PAPER

1.

STUDIES

ON THE

ABILITY OF MACROPHAGES

TO

PHAGOCYTISE PARTICLES OF DIFFERENT

SIZES

PAPER

2.

• • • • • • • •

THE EFFECT OF EXOGENOUS .A.DENOSINE TRIPHOSPHATE

ON THE PHAGOCYTIC ACTIVITY

OF MACROPHAGES

_{• • • • •} • •

PAPER

3.

STUDIES ON THE REQUIREMENTS

FOR THE

SPREADING

Page

1

29

45

OF PHAGOCYTIC CELLS ON A FLAT

SURFACE

•••••••••••

53

PAPER

4.

THE HYDROLYSIS OF EXOGENOUS ADENOSINE

TRIPHOSPHATE AND EXOGENOUS ADENOSINE

DIPHOSPHATE BY GUINEA

PIG PERITONEAL

MONONUCLEAR CELLS IN VITRO

_{• • • • • • • • •} • • • • • • • • • • • • •

PAPER

5.

92

THE SYNTHESIS OF ATP

BY CELLS

SPREADING

ON A

I

PAPER 6.

THE LOCALISATION BY ELECTRON MICROSCOPY OF NUCLEOSIDE PHOSPHATASE ACTIVITY IN

GUINEA PIG PHAGOCYTIC CELLS•••••••••••••••••••• 117

SECTION B: SOME MORPHOLOGICAL STUDIES OF

PHAGOCYTOSIS Al~D SOME CYTOCHEMICAL STUDIES OF POST-PHAGOCYTIC EVENTS

PAPER

7.

A MORPHOLOGICAL STUDY OF MACROPHAGES

SPREADING ON A FLAT SURFACE • • • • • • • • • • • • • • • • • • • •

PAPER

8.

BIREFRINGENCE PATTERNS IN GUINEA PIG

132

PERITONEAL MACROPHAGES ••••••••••••••••••••••••• 168

PAPER 9.

SOME MORPHOLOGICAL ASPECTS OF PHAGOCYTOSIS

BY

GUINEA PIG MACROPHAGES ••••••••••••••••••••••

173,b

PAPER 10.

AN ELECTRON MICROSCOPIC STUDY OF THE

DISTRIBUTION OF ACID PHOSPHATASE IN GUINEA PIG MACROPHAGES INGESTING FORMALIN-TREATED

SHEEP RED CELLS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

PAPER 11.

A COMBINED HISTOCHEMICAL AND ELECTRON

MICROSCOPIC STUDY OF CYTOPLASMIC DEGRANULATION IN PHAGOCYTISING GUINEA PIG POLYMORPHONUCLEAR

182

SUMMARY

AND

CONCLUSIONS

• • • • • • • • • • • • • • • • • • • • • • • •

REFERENCES

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

ACKNOWLEDGEMENTS

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

APPENDIX

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

Page

221

227

239

-I-INTRODUCTION

Metchnikoff introduced into biology the concept

that the ability of multicellular animals to resist colon-isation of their tissues by microorganisms, depends upon their possession of cells which are specialised to dis-criminate against, ingest and destroy invading microorga-nisms. Metchnikoff called these cells phagocytes from the

Greek translation of devouring cells. His original obser-vation that a splinter introduced into the tissues of a transparent starfish larva became surrounded by mobile cells, indicated to him the function of these cells and led to his cellular theory of immunity.

Metchnikoff summarised the most notable

characteris-tics of these cells when he wrote: "Phagocytosis is a phenomenon of considerable complexity. When it is

exhib-ited by leucocytes, these cells are in the first place affected by various substances which possess an attract-ion for them. They proceed towards these substances and then englobe them. Intracellular digestion may afterwards occur. Here then we have phenomena of sensibility, con-traction, ingestion and production of digestive fluidso"• This list of functions as noted by Metchnikoff suggests

2.

animals. It is therefore not surprising that phagocytic cells are regarded by some investigators as being primitive and as having retained the functions of acellular

ances-tral forms.

This thesis is mainly concerned with two of these functions: ingestion and intracellular digestion.

The process of phagocytosis

The term phagocytosis is used in this thesis to refer to the process which enables a cell to transport a relat-ively large particle (visible with the light microscope) from the extracellular space into its cytoplasm. Phago-cytosis begins after the cell makes contact with a suit-able particle and ends when the particle is completely surrounded by cytoplasm.

The literature on phagocytosis is voluminous and

despite a great many investigations, the mechanism respon-sible for phagocytosis has not been discovered. However, the literature reveals that phagocytosis by mammalian leu-cocytes is under the control of two main factors: metab-olic energy, and the forces of interaction between the surface of the cell and the surface of the particle to be phagocytised.

1. The surface tension theory for phagocytosis

at-This line of reasoning was taken up later by others

in-cluding Tait (1918-20) and Fenn (1921-22) who formulated

a theory for phagocytosis based on surface tension. Fenn

received the most recognition for his theory because it

was the most mathematically complete. According to Fenn,

phagocytosis can be explained in terms of the interaction

of forces which exist at three interfaces. In short, the

theory states that phagocytosis will occur after a cell

makes contact with a particle, provided that the surface tension between the particle and the suspending fluid is

greater than the sum of the surface tensions between the

particle and the cell, and the cell and the suspending

fluid. Therefore, according to this theory, any treatment

which increases the surface tension between the particle

and the suspending fluid will promote phagocytosis.

The surface tension theory of phagocytosis was

presen-ted at a time when other cellular phenomena were being ·

explained in terms of physics: Biochemistry was still in its infancy. Consequently, the surface tension theory

re-ceived a good deal of support and a substantial amount of

evidence was interpreted as being consistent with it.

Thus Mudd, McCutcheon and Lucke

(1934)

wrote: "The obvious

deductions from a theory which assigns to surface forces

4.

should be related in some orderly way with the surface properties of the particle phagocytised. This relation

has been verified over a considerable range of experimental conditions •••••••.• _•

_"

The important point about the

.,

surface tension theory is that it impl~s that the energy needed for phagocytosis is provided by "free" surface

energy, i.e., the cell does not supply the motive force but responds passively to forces of surface tension.

It is well to realise that the surface tension theory is based on the assumption that cells behave as perfect fluid spheres. The theory is therefore based on a model

system which bears very little resemblance to a living cell. In fact, the merits of the use of surface tension to explain phagocytosis have been discussed recently by Pethica (1961) who shows, on mathematical grounds, that the use of surface tension to explain contact equilibria is not permissible for cells. Furthermore, all of the evidence which is taken as being consistent with the sur-face tension theory is purely circumstantial. The surface tensions involved have not been measured, nor are there any methods available for measuring them (Pethica, 1961).

place between the surface of the cell and the surface of

the particle to be phagocytised are not known. Some of

the evidenc~ which indicates that surface factors play a

role in phagocytosis will now be considered.

2. The importance of serum opsonins

The realisation that the surface properties of a

par-ticle will determine whether or not the particle is

inges-ted by leucocytes was due to the simple yet crucial

ex-periments of Wright and Douglas (1904)0 These early

workers showed that mammalian leucocytes were not capable

of phagocytising certain bacteria unless the bacteria had

been first suspended in normal serum. Because the bacteria

had been washed free of serum before they were added to

the leucocytes, Wright and Douglas deduced that certain ·

components of the serum had coated the bacteria and made

them more susceptible to phagocytosis. They called these

components opsonins.

The findings of Wright and Douglas were important

because they showed that serum is able to promote

phago-cytosis by acting on the particles rather than by acting

on the cells. This knowledge helped to remove some of

the incompatibility which existed between Metchnikoff's

cellular theory of immunity and von Behring's hum.oral

theory of immunity. It had been shown earlier by Denys

60

bacteria much more rapidly in the presence of a specific

immune serum. Metchnikoff

(1894)

believed that such

ef-fects were due to the action of serum ("serum stimulins")

on the cells.

The term opsonin was used originally to describe

those components of normal serum which react with bacteria

to make them more susceptible to phagocytosis. Wright

and Douglas found that the opsonic activity of normal

serum was greatly reduced after the serum had been heated

to

55°c.

On the other hand, the opsonic activity of

spe-cific antisera appears to be relatively heat-stable. The

opsonins of immune serum were considered to be different

from specific antibody and were called tropins (Neufeld

and Rimpau,

1904).

The lrn.owledge that part of the opsonic activity of

normal serum is heat-labile provided the motive for a

large number of experiments which were designed to

deter-mine whether or not the heat-labile opsonic activity of

normal serum is identical with haemolytic complement.

The early literature on this topic was summarised by

Ecker, Weisberger and Pillemer

(1942)

and showed that there

was abundant evidence both for and against the

participa-tion of haemolytic complement in phagocytosis. According

to Gordon and Thompson

(1935)

the literature up until

1935

possibilities:

(a) Complement and normal opsonin are identical.

(b) Opsonin is a part of complement.

(c) Complement may when present in its complete

form act as an opsonin.

(d) The opsonin system is a different system to

complement but only able to act in the presence of

com-plement.

(e) Opsonin and complement are different systems

which act independently.

(f) Opsonin may be of the nature of an immune body

though thermolabile and non-specific.

Gordon and Thompson themselves provided evidence that

complement is not necessary for phagocytosis. More

recent-ly, Hirsch and Strauss

(1964)

published experimental re-sults which show also that beat-labile opsonins are not

identical with complement. Their interpretations were

based on the finding that opsonisation can take place in the absence of divalent cations whereas complement

fixa-tion requires divalent cafixa-tions.

A critical discussion of the role of haemolytic

com-plement in phagocytosis was presented recently by Boyden,

North and Faulkner,

(1965)

who pointed out that the

liter-atur contains too many conflicting reports to allow any

8.

of haemolytic complement in phagocytosis. Most of the

experiments which are interpreted as showing that either

complement or, some of its components function as opsonins,

are based on the assumption that the crude treatments

which are used to inactivate the components of complement

do not at the same time inactivate opsonins.

The knowledge that opsonins promoted phagocytosis by

modifying the surface of the particle to be phagocytised

is consistent with the surface tension theory of ·

phagocyt-osis by increasing the surface tension between the

part-icle and the suspending medium. This line of reasoning

led Mudd and his co-workers (Lucke, Mccutcheon, Strumia

and Mudd,

1929;

Mudd, Lucke, ~ Mccutcheon and Strumia,

1929)

to compare some of the surface properties of opsonised and

unopsonised gram-positive bacteria. They showed that serum

treated bacteria have increased cohesiveness, decreased

surface electric potential, decreased wetability by oil,

and are phagocytised at a much greater rate by leucocytes.

They showed also that specific antiserum is capable of

causing these changes at a much greater dilution than

normal serum. Similar experiments were performed with

protein "coated'' collodion particles. Opsonisation was

found to cause the same changes in surface properties of

1930).

In addition it was found that a specific

anti-serum against a soluble protein antigen increases greatly

the rate at which collodion particles, coated with this

protein, are phagocytised by leucocytes. This is good

evidence that the specific 6psonins of immune sera are

'

specific antibodies. Further evidence that specific

anti-bodies function as opsonins was supplied later by Ward

and Enders

(1933)

who showed that the opsonic activity of

a specific immune serum against a strain of pneumococci

can be blocked by treating the serum with a purified

pre-paration of the surface polysaccharide from this strain.

This evidence supported Zinssers

(1921)

unitarian concept

of antibodies. There is little doubt to-day that specific

antibodies (immune globulins) function as opsonins.

On the other hand, the molecules in normal serum

which function as opsonins have not been investigated

suf-ficiently to arrive at a general conclusion about their

nature. Tullis and Surgenor

(1956)

claim to have isolated

a "phagocytosis promoting fraction" from normal human

serum. The activity of the serum is said to reside in

theJ3-globulin fraction and promotes the phagocytosis of

starch particles by human leucocytes. However, it is not

known whether this fraction of serum contains opsonins

lOo

was not tested. It may have increased phagocytosis by

acting on the cells. Gabriel and Rosen (1963) found that the opsonins in normal human serum for certain

gram.-negative bacteria wereo(-macroglobulins. Turner and

Rowley (1963) arrived at a similar conclusion from a study

of the opsonins in pig serum.

3. The effect of surface-active agents I

The concept that phagocytosis was controlled by sur-·

face tension, motivated some workers to investigate the

effects on phagocytosis of substances which are known to

lower surface tension. Thus Berry and Spies (1949) claimed that decyl benzene sodium monosulphate, Triton-N-100,

Tween-20 and certain other detergents greatly enhance pha-gocytosis. They claimed also that pretreatment of bacteria

with dilute solutions of detergents depressed the rate at

which bacteria were phagocytised by leucocytes: a result consistent with the surface tension theoryo In contrast,

Nungester and Ames (1952) investigated the effect on

pha-gocytosis of a large number of surface active agents and concluded that there was no relationship between lowering

of surface tension and phagocytosis. Nungester and Ames

point out that the results obtained with detergents are

hard to interpret because detergents have drastic effects on biological molecules. For instance, they are potent

It is therefore surprising that those who claim that

de-tergents enhance phagocytosis by altering surface tension,

n

do not cdsider the possibility that their results may

have been due to the denaturation of protein on either the

surface of the cell or, the surface of the bacteria.

4.

The significance of surface charge

It was shown in a series of experiments by Mudd and

co-workers (reviewed by Mudd, Mccutcheon and Lucke,

1934)

that treatment of bacteria and of protein coated collodion

particles with either normal or specific immune serum

de-creased th-e electrophoretd.c mobility of these particles.

It was -found that specific immune serum was capable of

decreasing the surface electric charge at much greater

di-lutions than was normal serum. Similar findings were

pu-blished earlier by Northrop and De Kruif

(1921).

Mudd

and his co-workers interpreted this information as

sup-porting the surface tension theory because it indicated

that opsonisation caused changes in the surface properties

of the particles to be phagocytised. The ability of the

opsonins of both normal and specific immune sera to lower

the surface charge on bacteria was studied again by

Nungester, Ames and Lanning (1952) who confirmed the

re-sults of the earlier investigators. However, Nungester

et al were more aware of the significance of their findings

and pointed out that both the leucocytes and the

12.

teria displayed a nett negative charge. They pointed out

also that although specific immune serum can significantly

reduce the charge on bacteria, the bacteria nevertheless

remain negatively charged: an interesting situation since

bodies of like charge repel one another. They suggested

therefore, that the surface of the cell may contain regions

of positive charge and regions of negative charge; oth

r-wise the bacteria could not be approached by the cell.

There appears to be little doubt that mammalian

leu-cocytes (Bangham, Seaman and Pethica,

1958)

and indeed all

cells in suspension carry a nett negative charge (Pethica,

1961).

The question therefore arises as to how it is

pos-sible for a negatively charged cell to approach close

enough to a negatively charged particle in order to ingest

it. This problem has been discussed recently by Pethica

(1961)

in relation to cellular adhesion. Pethica shows,

on theoretical grounds, that it is indeed possible for two

negatively charged cells to overcome the force of repulsion

and make contact, provided that the magnitude of the

sur-face charge on each is different, and provided that the

cells approach one another with parts of their surface

mem-branes having radii of curvatures of O.l microns or less.

Having overcome the force of repulsion the cells will then

adhere because of short range forces of attraction, i.e.,

a force of repulsion at long range and a force of

at-· traction at short range. It is therefore easy to imagine that a phagocytic cell may be able to make contact with negatively charged particles by sending put thin narrow processes or pseudopods.

In the light of this knowledge, it is possible that the function of opsonins is to ensure that the surface charge on the particle to be phagocytised is lower than the surface charge on the leucocyte.

5. The influence ·of pH

If surface charge is important in phagocytosis then it might be suspected that phagocytosis would be sensitive to changes in pH. This appears not to be the case. It has been shown by Tucker, Hill and Gifford

(1963)

that

the phagocytosis of mycobacteria by rabbit mononuclear phagocytes shows little variation between pH

6.5

and pH 9. Furthermore, phagocytosis takes place on either side of this pH range. Hirsch and Strauss

(1963)

also noted that the phagocytosis of bacteria by rabbit poly-morphonuclear leucocytes was not affected by varying the pH between 6 and _,,

8.

These results confirmed the earlier results of Evans (1922).

On the other hand, the pinocytosis of ferritin by amoebae does appear to depend on surface charge.

14.

ferritin to the surface of amoebae can be prevented by

changing the charge on the ferritin molecule and by

changing the pH of the medium. They concluded that the

binding off rritin to the surface of amoebae takes place

when the ferritin is positively charged. However, despite

these results, it would be unwise to generalise about

phagocytosis from studies of pinocytosis in acellular

ani-mals. It is possible that pinocytosis and phagocytosis

are different m~chanisms. Furthermore, free-living

acell-ular animals have evolved mechanisms which enable them to

exist under large fluctuations of their environment.

Again, the surfaces of amoebae are structurally specialised

(Brandt and Pappas,

1960).

6. The role of Calcium and Magnesium

There is a large number of compounds and ions which

influence the phagocytic activity of mammalian leucocytes

(Mudd, McCutcheon and Lucke,

1934;

Berry and Spies,

1949).

The early literature dealing with the effect of calcium

was reviewed by Mudd, McCutcheon and Lucke

(1934)

and

shows that calcium ions are necessary for efficient

pha-gocytosis. More recent experiments· on the role of calcium

and magnesium ions were carried out by Wilkins and Bangham.

(1964).

These workers tested the hypothesis that calcium

ions act ·by reducing the surface charge on the particle

starch particles and polystyrene particles _{by rabbit}

polymorphonuclear leucocytes _{and found that maximum}

pha-gocytosis occurred when the concentrations _{of calcium and}

magnesium ions were 10-3 M, i.e., at the _{concentration of}

calcium ions in bloodo However, _{at this concentration of}

calcium, the polystyrene _{particles had maximum}

electro-phoretic mobility. On _{the other hand, starch particles}

displayed zero electrophoretic mobility at all concentra-tiohs of calcium yet, were phagocytised _{most rapidly when}

the concentration of calcium was 10-3 M. _{Wilkins and} Bangham concluded that calcium ions probably function by cross-linking serum proteins between the cells and part-icles, i.e., _{by forming calcium-bridges. However, their}

finding that barium ions inhibit phagocytosis at all con-centrations would seem to indicate _{that the function of}

calcium ions is not to cross-link the _{proteins on the} sur-faces of the cells with those on the _{surfaces of the} part-icleso _{Barium. ions should be equally capable of combining}

with the negatively charged groups of the _{proteins on the} surfaces of cells and particles. In fact, the inability of barium ions to substituze _{for calcium ions indicates}

that calcium ions may be _{needed or enzymatic reactions}

at the surface of the cell. It _{is well known that the} activities of many enzymatically controlled reactions

16.

Further knowledge about the role of calcium ions in

phagocytosis was furnished by Hirsch and Strauss

(1963)

who showed that although calcium ions are necessary for

phagocytosis, they are not necessary for the opsonisation

of bacteria by normal serum. In other words, calcium ions

exert their influence on phagocytosis after the particles

have been prepared for phagocytosis by serum opsonins.

The above discussion shows that despite the wealth

of descriptive information about the role of opsonins in

phagocytosis, the reason why opsonins are able to increase

the rate of phagocytosis is not known. Attempts to

ex-plain phagocytosis as resulting from forces of interaction

between the surface. of the cell and particle appear so

far to have been unp~ofitable. More profitable experiments

probably will be based on a better knowledge of the

mole-cular make-up of the plasma membrane of phagocytic cells.

The role of metabolic energy

In

1933

Baldridge and Gerard showed that the addition

of bacteria to a suspension of dog leucocytes caused a

marked increase in the rate at which the leucocytes

con-sumed oxygen. Because the leucocytes had phagocytised

the bacteria, Baldridge and Gerard concluded that the

in-creased oxygen consumption was causally related to

phago-cytosis. They stated: 0 _{As other cells increase their}

that the respiration of leucocytes would increase during

active phagocytic ingestion - unless the entire process

is controlled by physical tension".

Judging from the literature, the discovery that

pha-gocytosis is associated with an increased rate of energy

metabolism did not influence the then generally accepted

view that the energy for phagocytosis was supplied by

sur-face tension. Berry and Spies

(1949)

in their review

article on phagocytosis gave very little mention to

meta-bolic energy and stressed the evidence in favour of the

surface tension theory. Woodin

(1963)

has expressed the

view that the changes in cellular metabolism during

pha-gocytosis are related to post_:phagocytic events.

The experim ntal results of Baldridge and Gerard have

been confirmed on many occasions (Delauny, Pages and

Maurin,

1946;

Stahelin, Suter and Karnovsky,

1956;

Becker, Munder and Fischer,

1958;

Sbarra and Karnovsky,

1959;

Cohn and Morse,

1960;

Straus and Stetson,

1960;

Iyer, Islam and Quastel,

1961;

Rossi and Zatti,

1964,

and

others). At the present time, most of the experimental

effort is devoted to discovering the details of them

ta-bolic changes associated with phagocytic activity.

Experiments with metabolic inhibitors (Sbarra and

Karnovsky,

1959;

Cohn and Morse,

1960;

Iyer, Islam and

180

Saito, Milofsky and Karnovsky, 1963) indicate that the

energy needed for phagocytosis is derived from anaerobic

glycolysis. Thus, classical inhibitors of anaerobic

gly-colysis such as sodium fluoride and iodoacetate inhibit

both phagocytosis and glycolysis in phagocytic cells.

In contrast the phagocytic activity of most mammalian

pha-gocytic cells is not affected by compounds such as

potas-sium cyanide, 2,4-dinitrophenol and antimycin-A which

pre-vent the synthesis of adenosine triphosphate by the

res-piratory chain. Furthermore, most phagocytic cells

dis-play a high residual respiration in the presence of

pot-assium. cyanide and this respiration is increased during

phagocytosis -(Becker, Munder and Fischer, 1958; Oren,

Farnham, Saito, Milofsky and Karnovsky, 1963). In other

words, the increased oxygen consumption during phagocytosis

is not linked to the cytochrome system.

It was shown by Sbarra and Karnovsky (1959) that

pha-gocytosis by polymorphonuclear leucocytes is associated

with a large increase in the rate at which glucose is

oxi-dised via the hexose monophosphate shunt pathway (an

alter-nate pathway for glucose oxidation). This finding was

confirmed later by others (Iyer, Islam and Quastel, 1961;

Oren, Farnham, Saito, Milofsky and Karnovsky, 1963;

Rossi and Zatti, 1964). Thus, although the energy for

phagocytosis appears to come from anaerobic glycolysis

phagocytosis is nevertheless associated with an increased

activity of the alte,rnate pathway for gluoose oxidation

and with an increase in respiratory activity which is not

dependent on the cytochrome system. To explain these

findings Evans and Karnovsky

(1961)

postulated that two

key enzymes are involved, and offered the following

hypo-thesis: During phagocytosis, a cyanide-insensitive

oxi-dase is activated. This oxidase preferentially oxidises

reduced diphosphopyridine nucleotide (NADH) which accounts

for the increased respiration. Some pyruvate from

gly-colysis is then in excess of the diphosphopyridine

nucleo-tide (NAD) available to convert it to lactate. A lactate

dehydrogenase which is linked to reduced

triphospho-pyridine nucleotide (NADPH) catalyses the conversion of

this pyruvate to lactate and this reaction increases the

availability of triphosphopyridine nucleotide (NADP).

NADP has been shown to be a rate limiting factor for the

hexose monophosphate shunt (Beck,

1958).

The

cyanide-insensitive oxidase was supposed to be released from the

specific granules of polymorphonuclear leucocytes during

phagocytosis (Evans and Karnovsky, 1961, 1962). This

view recently has been changed because it was found by

Cagan and Karnovsky

(1964)

that the cyanide-insensitive

oxidase is in the cytoplasm of polymorphonuclear leucocytes.

20.

evidence which shows that the NADH-oxidase is indeed

associated with the cytoplasmic granules but is not libe-rated during phagocytosis.

On the other hand, Roberts and Quastel

(1964)

explain the increased oxygen consumption during phagocytosis as being due to the liberation from the cytoplasmic granules of peroxidase which oxidises NADPH immediately. This

would explain the increased oxygen consumption and the in-creased activity of the hexose monophosphate shunt. It is known that perox.idase is capable of catalysing the oxidation of NADPH (Akazawa and Conn,

1958).

It would

appear in fact, that both enzymes are involved in the meta-bolic changes associated with phagocytosis (Rossi and

Zatti,

1964;

Cagan and Karnovsky,

1964).

Despite the accumulation of detailed knowledge about the changes in cellular metabolism associated with phago-oytosis, the significance of the increased oxygen consump-tion and the significance of the acti'vation of the hexose monophosphate shunt are not known. Karnovsky

(1961, 1962)

has proposed that the increased activity of the hexose

required for phagocytosis, it would seem that the increase

in oxygen consumption and the increased activity of. the

shunt are concomitant events and are not essential {at

least in the short run) to the mechanism of phagocytosiso

The l _i terature cited above deals mainly with

phago-cytising polymorphonuclear leucocytes. Information

con-cerning the metabolism of phagocytising mammalian

mono-nuclear phagocytes is not nearly so detailed. Oren,

Farnham, Saito, Milofsky and Karnovsky

(1963)

have shown

that phagocytosis by guinea pig peritoneal macrophages .

is associated with an increased oxygen consumption which

is insensitive to cyanide, and with an increase in the

activity of the hexose monophosphate shunt. They showed

also, by the use of metabolic inhibitors, that the energy

for phagocytosis is probably derived from anaerobic

gly-colysis. On the other hand, the energy required for

pha-gocytosis by guinea pig alveolar macrophages is derived

from cytochrome-linked respiration • . Different results

were obtained by Ouchi, Selvara and Sbarra

(1965)

who

studied phagocytising rabbit alveolar macrophages. They

found that phagocytosis by rabbit alveolar macrophages is

not blocked by either those inhibitors which block the

cytochrome system or, by those inhibitors which block

glycolysis. These workers did not however, venture an

22.

One of the most striking features of the increased

respiration associated with phagoeytosis is that it

oc-curs rapidly, but lasts for a short time only. This

find-ing has been noted by most investigators but was shown

very convincingly by Rossi and Zatti

(1966)

who measured

changes in oxygen tension by a sensitive polarographic

technique. They found that oxygen consumption by

polymor-phonuclear leucocytes increases within 60 seconds after

adding particles. They interpreted this evidence as

in-dicating that oxygen consumption increases before the

spe-cific granules of polymorphonuclear leucocytes lyse and

before lactic acid accumulates. This evidence tends to

disprove the original hyp0thesis of Evans and Karnovsky

(1961)

that phagocytosis gives rise to an accumulation of

lactic acid which lowers the intracellular pH, and causes

the specific granules to release the oxidase responsible

for increased oxygen consumption. The experiments of

Rossi and Zatti indicate also that the increase in energy '

metabolism associated with phagocytosis occurs immediately

after the cells make contact with the particles, i.e.,

before any particles are ingested. This counters attempts

to explain the increase in energy metabolism as being

solely due to post-phagocytic events (Woodin,

1963).

A survey of the literature on phagocytosis shows that

ways in which phagocytosis can be modified, the

mecha-nism responsible for phagocytosis ·is unknown.

Further-more, although the metabolic studies on phagocytising

cells have revealed that phagocytosis requires a source

of metabolic energy, they give no information about the

actual mechanism of phagocytosis, except that it utilises

adenosine triphosphate. It would be wrong to disregard

the importance of surface interactions in the process of

phagocytosis. The evidence is still consistent with the

hypothesis that the motive force for phagocytosis is

gen-erated by surface interactions (although not by surface

tension), and that metabolic energy is needed to produce

changes in the consistency of the cytoplasm in the

vicin-ity of the particle being phagocytised. It was suggested

many years ago by Loeb (1921) that the cell would need to

decrease the viscosity of its cytoplasm in order to ingest

particulate matter.

Post-phagocytic events

Metchnikoff was aware that phagocytio cells possess

mechanisms which enable them to digest ingested

micro-organisms. The existence of such mechanisms was a

pre-requisite for his cellular theory of immunity. Metchnikoff

(1905) conceived that phagocytic cells secrete digestive

ferments around ingested microorganisms. He refered to

24.

the cells would need to possess mechanisms to protect

themselves against their own cytases.

There was very little knowledge about the hydrolytic

enzymes of cells until de Duve and his co-workers (de Duve,

Pressman, Gianetto, Wattiaux and Appelmans,

1955)

isol-ated from liver a cellular fraction which contained most of the cellular acid hydrolases. This fraction was

sub-sequently shown to be composed of membrane limited

gran-ules with a mean diameter of 0.41 microns (Novikoff,

Beaufay and de Duve,

1956).

These granules were called lysosomes and were given the status of cellular organelles

(de Duve,

1959).

The opinion was expressed by de Duve at al

(1955),

that lysosomes are probably concerned with localised

intra-cellular digestion. Evidence for this idea was supplied

by Rose

(1957)

who observed with phase contrast micros-copy that newly formed pinocytotic vesicles in HeLa cells

fused with pre-existing dense granules. Rose suggested that the dense granules are probably lysosomes and that

they secrete digestive enzymes into pinocytotic vesicles.

Further evidence for this idea was furnished by Straus

(1958),

who showed by histochemical -methods, that vacuoles

containing ingested foreign protein acquire lysosomal

enzymes. Lysosomes have been found in many different types

The importance of lysosomes in mammalian phagocytic

cells was recognised by Hirsch and Cohn (1960) who

con-firmed the early observations of Robineaux and Frederic

(1955) that phagocytosis by polymorphonuclear leucocytes

is associated with the disappea-rance of the cytoplasmic granules of these cells. Hirsch and Cohn (1960) noticed

that specific granules lyse in the vicinity of ingested material and suggested that cytoplasmic degranulation in

phagocytising polymorphonuclear leucocytes is concerned with intracellular digestion. These observations were

followed by an investigation of the distribution of

lys-osomal enzymes of "resting" and phagocytising

polymor-phonuclear leucocytes (Cohn and Hirsch, 1960; Hirsch

and Cohn, 1960). It was found that in "resting"

poly-morphonuclear leucocytes-, the cellular acid hydrolases

are associated with the granule fraction, whereas the acid

hydrolases in activ~ly phagocytising cells are in the

supernatant fraction. The results were interpreted as

meaning that the granule-associated hydrolases are

"released" during phagocytosis. Cinemicrophotographic

evidence for this concept was furnished by Hirsch (1962)

and by Archer and Hirsch

(1963)

and electron microscopic

evidence was furnished by Lockwood and Allison

(1963),

and by Zucker-Franklin and Hirsch (1964).

hydrolases of mammalian macrophages (Cohn and Wiener,

1963, 1963b), and the results of these studies led to

es-sentially the same conclusiono Electron microscopic

evi-dence for the concept that the lysosomes of macrophages

fuse with vacuoles containing ingested bacteria was

pre-sented by North and Mackaness (1963)0

Although there is no doubt that phagocytic cells

possess the enzymes necessary for intracellular digestion

it was not until fairly recently that Cohn (1963)

inves-tigated the actual degradation of ingested bacteria by

phagocytic cells. By using isotopically labelled bacteria

he was able to show that most of the labelled molecules

from digested bacteria are excreted by the cell, and that

some of the p3 2 label is incorporated into cellular lipid.

He showed also, that heat-killed bacteria are degraded

much more readily than living bacteriao

Hirsch, (1956) has expressed some doubt about the

ability of intracellular digestive enzymes to kill ingested

bacteria. He points out that it is possible that

digest-ive enzymes act only after the bacteria have been killed,

and that the actual killing of bacteria may be due to a

combination of the actions of acid and bacteriocidal

sub-stances. It was shown by Rous (1925) that cytoplasms

imm diately surrounding ingested bacteria, may have a pH

accumulate lactic acid during phagocytosis (Becker,

Munder and Fischer, 1958; Sbarra and Karnovsky, 1959; Cohn and Morse, 1960; Rossi and Zatti, 1964).

Further-more, Dubos (1953) has_ demonstrated that lactic acid can be bacteriocidal at low pH.

Two well documented bacteriocidal substances which are lrnown to be possessed by mammalian phagocytic· cells are lysosyme and phagocytin. Lysosyme is capable of

kill-ing certain gram-positive organisms (Flemi~g, 1922;

Hiatt, Flood, Karush and Engle, 1952; Myrvi k and Wiesser,

1955; Hirsch, 1960). Phagocytin is present in

polymor-phonuclear leucocytes and is active against a range of gram-negative organisms (Hirsch, 1960).

In addition to cellular bacteriocidal substances,

there are substances in blood such as haemolytic

comple-ment and properdin (Pillemer, Blum, Lepow, Ross, Todd and

Wardlaw, 1954), which may also contribute to the

degra-dation of microorganisms before they are phagocytised.

On the other hand, opsonins do not appear to contribute to the intracellular digestion of microorganisms. On the contrary, bacteria which have been pretreated with a spe-cific antiserum are degraded much more slowly inside pha-gocytic cells (Cohn,- 1963b).

It is clear that the exact sequence of events which

280

SOME EXPERIMENTS CONCERNED

WITH THE

29.

PAPER

1.

STUDIES ON THE ABILITY OF MACROPHAGES TO

PHAGOCYTISE

PARTICLES OF DIFFERENT

SIZES

The phagocytosis of a particle by a phagocytic cell results in the enclosure of the particle in a membrane-limited phagocytic vacuole. (Essner, 1960; Journey and Amos, 1962; Karsbad, Kessel, de Petris and Monaco, 1964; North and Mackaness, 1963a, b; Pearson, Freeman and

Hines, 1963; Leake and Myrvik, 1966). The way in which the phagocytic vacuole is formed appears to have been de-scribed best by Essner (1960) who studied the phagocytosis of red cells by peritoneal macrophages. According to

Essner, a macrophage ingest~ a red cell by sending out an enveloping process which progressively surrounds the red cell on all sides. In this way the red cell is enclosed within a vacuole the membrane of which is derived from the plasma membrane of the macrophage. Therefore each time a particle is ingested, the cell internalises part of its plasma membrane. The area of plasma membrane internalised cannot be less than the surface area of the particle.

The aim of the experiments in this paper was to deter-mine whether or not the availability of plasma membrane

is a limiting factor in phagocytosis. This idea can be

icles macrophages are capable of ingesting. If the avail-ability of plasma membrane is a limiting factor, then

the cells should cease ingesting each type of particle

when the same amount of surface area has been internalised. Materials and Methods

Animals: Heterogenous groups o·f male guinea pigs were used. They were supplied by The Animal Breeding

Establishment, John Curtin School of Medical Research.

Phagocytic cells: Mononuclear phagocytic cells (macropha-ges) were obtained from the peritoneal cavity. Each ani-mal was killed, the abdominal skin was removed and the

peritoneal cavity was injected with 20 mls of Hanks' solu-tion containing 10 international units of heparin per ml. The Hanks' solution was injected with a sterile needle and syringe and the peritoneal washings were withdrawn

into the same syringe. The cell content of the peritoneal washings was determined with a Spencer 0 _Bright-line11 hae-mocytometer. Hanks' solution was added to the peritoneal washings to give 5 x 105 cells per ml. Preparations ob-tained thus from several animals at a time were pooled before use.

Test particles: These were formalin-fixed guinea pig red cells, formalin-fixed sheep red cells, formalin-fixed

yeast cells, and three sizes of polystyrene spheres.

31.

by cardiac puncture which was prevented from clotting by

adding it to

3

volumes of a solution containing

0.8

per

cent sodium citrate and 0.42 per cent sodium chloride.

The red cells were separated and washed three times by

centrifugation and resuspension in a solution of 0.9 per

cent sodium chloride. They were centrifuged finally into

a pellet and fixed with

3

volumes of 10 per cent

formal-dehyde in

0.9

per cent sodium chloride. Fixation took

place for 24 hours at

4°c.

The cells were then washed

three times in distilled water and transferred to dialysis

bags and dialysed against running tap water for 72 hours.

They were then washed three times in a solution of Oo9 per

cent sodium chloride and finally stored in the sodium

chlo-ride solution as a

30

per cent suspension at

4°c.

Sheep red cells were separated from citrated blood

obtained from the local abattoir and were prepared in the

same way as the guinea pig red cellso

Yeast cells (Saccharomyces cerevisiae) were purchased

from a local bakery. They were washed three times in

dis-tilled water and then treated in the same way as the red

cells.

Polystyrene spheres were supplied by the Dow Chemical

Co., Michigano They were dialysed against large volumes

of distilled water for

48

hourso

-of the three sizes of polystyrene spheres were given by the manufacturers. Their mean diameters ·were Oe50,

o.81

and

1.28

microns respectiyely.

The diameters-of the formalin-fixed red cells and yeast cells were measured with a calibrated measuring eye piece utilising bright field microscopy and a ·magnification of x 1000. The diameters of _50 cells of each type were

measured and means and standard deviations were calculatedo The volumes and surface areas of the polystyrene

spheres were calculated by using the formulae for the vo-lmne and surface area of a sphere. The volume and surface area of yeast cells W~iecalculated in the same way. The mean volume of red cells was obtained from a knowledge of ~ the total volume occupied by a known number of packed red cells. The red cells were centrifuged in haematocrit

tubes for 1 hour at 3000 r.p.m. and the volume occupied by packed red cells was read from the scale on the tubes.

To obtain the surface area of red cells, the red cells were assumed to be flat cylinders. The surface area of a cylinder can be calculated if its height and diameter are known. The height of red cells was obtained by sub-stituting the values for their diameter and volume in the formula for the volume of a cylinder.

Culture chambers: Each culture chamber consisted of a

/

33.

18

mm

in diameter bored through it. The hole was covered

on one side by a coverslip which was attached to the

Perspex slide by a mixture consisting of one part

petro-leum jelly and one part paraffin wax. This coverslip

served as the floor of the chamber so formed.

Coverslips were cleaned by boiling them for 30 minutes

in dilute hydrochloric acid and then in three changes of

distilled water. They were dried individually in racks

' '

in an oven at 110°c.

Perspex slides were cleaned by boiling them in dilute

detergent and then washing them in several large volumes

I

of distilled water. They were dried in an oven at 110°c.

Each culture chamber was kept in a petri dish and

the entire unit was sterilised by placi~g it in an oven

at 110°c for 6 hours. ' This .procedure remelted tbe wax

mixture which was retained between the Per,sp~x and

cover-slip by capillarity.

I

Serum: Serum was prepared from guinea pig blood which

was obtained by cardiac puncture. The blood was placed

in sterile test tubes and allowed to clot for 1 hour at

37°c.

The test tubes were then placed in a cold room at

4°c

for 3 hours to retract the clot. The serum was

trans-fered to test tubes and centrifuged at 3000 r.p.m. to

remove particulate matter. The sera from no less than

volumes at

20°c.

Culture medium: The medium for maintaining cells in vitro

consisted of 20 per cent guinea pig serum dissolved in

Hanks' solution. The medium was adjusted to pH 7 with

sodium bicarbonate immediatley before _use. The

compos-ition of Hanks' solution is given in Appendix 1.

Phagocytosis:

0.8

mls of the cell suspension obtained

from the peritoneal cavity of guinea pigs (5 x 105 cells

per ml.) was added to each of a number of culture ·chambers.

The chambers were placed in an incubator and the cells

were allowed to settle and spread out on the floors of

the chambers at

37°c

in an atmosphere consisting of

5

per

cent carbon dioxide in air. The chambers were then

washed out three times with Hanks' solution and the cells

were allowed to remain in a final change of Hanks'

solut-ion until the particles were added.

The particles were suspended in Hanks' solution

con-taining 20 per cent serum. They were present in the

fol-lowing numbers per ml.: 10

8

guinea pig red cells, 10

8

sheep red cells, 108 yeast cells and 109 of each of the

polystyrene spheres.

008

mls of one or the other of the

particle suspensions was added to each of a number of

cult-ure chambers. The chambers were then placed in an

incub-ator and phagocytosis was allowed to proceed at

37°c

in an

atmosphere consisting of 5 per cent carbon dioxide in

35.

At different time intervals one or more chambers were

removed to assess phagocytosis. The phagocytosis of each

type of particle was studied separately 10 times.

In order to determine the number of particles ingested

by macrophages adhering to the floors of the chambers,

it was necessary to remove all particles which had not

been ingested. This was accomplished by inverting the

chambers over a sink and squirting the cells with a firm

jet of Hanks' solution delivered from a plastic wash

bottle. About 10 mls of Hanks' solution was used for each

chamber. This procedure removed those particles not

in-gested but left the macrophages intact on the coverslips.

Chambers were then filled with a solution containing 10

per cent formaldehyde and Oo9 per cent sodium chloride.

The cells were allowed to remain in this solution until

the experiment was terminated. Chambers were sealed

with a coverslip and inverted on the stage of a microscope.

The number of particles contained by 100 macrophages and

the percentage of macrophages containing particles were

assessed.

Results

The design of the experiments ensured that every cell

had an equal opportunity to phagocytise particleso The

particles quickly formed _{several layers on top of the cells}

,--)(

U) w (.9 ₁₅

<l:

I a..

0 er u

<l:

'.L 10

0 0

,--er w Q_ U) w 5

...J

u i= er

~

d z

0 25, 50 _{75 100 125 150 175 200 225}

TIME IN MINUTES

Figure 1.

Phagocytosis of formalin-fixed red cells

by guinea pig macrophages . The mean results of

10 experiments. Standard deviations are shown as

36.

taken by another particle. This ensured that changes in

the phagocytic activity of the cells were not due to

changes in the availability of particles. There were at

least 1000 times more particles than cellso This meant

that there were many more particles present than the cells

were capable of ingesting.

The cell population adhering to the floors of the

chambers consisted of over 97 per cent mononuclear

phago-cytes. Invariably, all of these cells were phagocytising

within 10 minutes of adding the particles.

The phagocytosis of formalin-fixed sheep red cells

The phagocytosis of sheep red cells is described

first in order to illustrate the way in which most

part-icles were ingested. The results of 10 experiments are

shown in figure 1, where it will be seen that the graph

for phagocytosis is step-like. The cells phagocytised

particles at a constant rate, and then ceased

phagocytis-ing for 25-30 minutes after which they again began

phago-cytising particles and continued to phagocytise until they

became saturated. The cells were considered to be

satur-ated because further phagocytosis could not be detected

over a further period of 2 hours. In fact, there was a

slight reduction in the numbers of formalin-fixed sheep

red cells contained by the macrophages after saturation.

However, formalin-fixed red cells and formalin-fixed

yeast cells were highly resistant to intracellular

digest-ion. This was investigated by allowing macrophages on

glass to ingest a small number either red cells

or

yeast

cells and comparing the number of these particles visible

in macrophages before and after the preparation had been

incubated at

37°c

for 2 hours. There was a reduction of

not more than 5 per cent in the number of particles

con-tained by macrophages after 2 hours incubation. The phagocytosis of particles of different sizes

The graphs representing the phagocytosis of particles

of different sizes are shown in figure 2. It will be

seen that the form of all of the graphs except one, is

similar: phagocytosis was interrupted by a period during

which little or no phagocytosis could be detected. This

will be referred to as the "rest period". On the other

hand, the phagocytosis of the 0.81,,U diameter polystyrene

spheres took place without interruption until the

macro-phages became saturated.

It will be seen from figure 2, that macrophages were

capable of ingesting more smaller particles than larger

ones. However, the rate of phagocytosis was not related

to the size of the particles. Yeast cells were ingested

at a much faster rate than sheep red cells although both

Figure 2.

The ingestion of 0.81f{diameter polystyrene

spheres

(A),

l.28~{diameter polystyrene spheres

(B),

3.68);/diameter of yeast cells

(C),

3.9f1diameter

sheep red cells

(D),

6.3)t diameter guinea pig red

cells

(R).

The ingestion of each type of

part-icle was studied 10 times. Vertical lines represent

C\J ID r -x (.f) w (9 <l I Q.. 0 0:: u <t: L 0 0 r-0:: w Cl. (f) w _J u t-o::

~

.

0 z 50 40 30 20 10 B C D

V

E

(,I

..

0 50 100 150 200 250

TIME IN MINUTES

Particle and No. ingested No. ingested

diameter in at "rest period" at saturation

microns _{* *}

Guinea pig red cells

6.3 ! 0.58 4.0 1.2

Sheep red cells

3.9 ! 0.09 9.3 15. 7

-

--Yee.st cells

+

3.68 - 0.29 11.1 19

Polystyrene spheres

1.28 ! 0.202 23.3 39.6

Polystyrene spheres

0.81 60.3

*

The numbers of particles ingested at the

"rest ueriod" and at saturation were taken from

the results ~resented in figure 2.

Table 1.

Sum of diemeters et

"rest period" LI..

25.2

36.3

40.8

29.1 no "rest

period"

SUJTl of SUP.I of Swr. of Sum of

di11meters nt vol umfrn n t surfEce nreas surface ereps setu..rrttion saturr.tion at "rest Pet"iod" at setLlt"ation

JJ ,AL"! AL z ,i.1}·

45. 4 224.4 577.6 1040. 2

-

-60.2 187.ll5 _--- 308.1 _- 502. 6_{- - -}· _{- - ·}

6g.9 _{503.7 472.5 807.5}

4-9.5 4 3. 2 114 .1 194. 0

no "rF?st

The aim of this study was to determine whether or not

macrophages would cease ingesting each type of particle

when a definite amount of surface area had been ingested.

In other words, the mean surface area of each type of

part-icle multiplied by the number ingested should be a

con-stant.

The relationship between the surface areas of the

particles and the number ingested is shown in table 1.

This table shows also the relationship between the

dia-meters of the particles and the number ingested, and the

relationship between the volumes and the number ingested.

It will be seen that the phagocytosis of each type of

\

particle did not involve the same amount of surface area.

The total surface area involved in the phagocytosis of

guinea pig red cells was 10 times more than that involved

in the phagocytosis of o.8l4'diameter polystyrene spheres.

Likewise, the total volumes involved in the phagocytosis

of each type of particle were not equal. On the other

hand, table 1 indicates that the number of particles

in-gested may have been related to the diameters of the

part-icles. Thus the sums of the diameters of each type of

particle ingested at saturation were of the same

magni-tude and varied between

45.4

and

69.9

microns. This

de-gree of variation would be expected because the

Table 2

The relationship between the diameter of particles

and

the number ingested at saturation.

A

result

ob-tained with a single population of cells.

Particle and mean diameter in

microns Guinea pig red

cells

6.3

:!:

0.58

Mean number ingested per

macropp.§.ge

10.6

-Sheep red cells +

3.9 -

0.09

20.2

Yeast cells

3.68

_-

:!:

0.29

_{- -}

___

..._

___ _

19.9

Polystyrene spheres

1.28

:!:

0.20

Polystyrene spheres

Oo81

Polystyrene spheres

0.5

69.1

115.3

196.5

Sum of diameters in

It will also be seen from Table 1, that the number of particles ingested at the urest period" also was not related to either the surface areas or, the volumes of the particles. The numbers of particles which were in-gested at the rest period ·appeared to be related to the diameters of the particles.

The relationship between the diameter of the particles and the number of particles ingested was investigated

further by comparing the number of particles of different sizes ingested by a single population of cells. This was done by adding a suspension of each type of particle to two chambers and counting the numbers of each type of particle ingested by 100 macrophages after 3 hours at

37°-c. The results of one experiment are shown in Table 2, where it will be seen that the sums of the diameters of each type of particle ingested

at

saturation, fall be-tween

98.2

microns (for the smallest particle) and

66.8

microns (for the largest particle). Discussion