Cytological studies of ovine alveolar macrophages : interaction with Mycoplasma ovipneumoniae in vitro : this thesis is presented in partial fulfilment (30%) of the requirements for the degree of Master of Philosophy in Veterinary Pathology at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

CYTOLOGICAL STUDIES

OF OVINE ALVEOLAR MACROPHAGES: INTERACTION WITH

MYCOPLASMA OVIPNEUMONIAE IN VITRO

)

Thi s thesis is presented in par t ial fulfilment (30%) o f the requiremen ts for the degree o f Master o f Ph ilosophy in Veterinary Pa thology a t Massey Universit y .

AYAn ^AL.KAISSI

1981

ABSTRACT

The attachment between Mycopl a sma o vipneumoniae organisms and ovine alveolar macrophages was studied in culture for a

24

hour period and antibody-med iated phagocytosis of M. ovipneumonia e organisms was observed b y both scanning and transmission electron microscopy. Mycopl a sma o vipneumoniae o rganisms have the ability to attach to the alveolar macrophage membrane without inducing phagocytosis although they stimulated mitotic division in early cultured cells. The add ition of specific antibody to the

mycoplasma-macrophage cultures provoked phagocytosis of surface attached and surround ing M . ovipneumoni a e organisms. Alveolar macrciphages stimulated by specific antibody showed rapid and

extensive spreading on the glass coverslip and prominent membrane ruffl ing and filopodia. Many exterior openings and fine cytoplasmic pits were also evident which may represent pinocytotic vesicle

formation sites . With transmission electron microscopy M . ovipneumoni a e organisms were observed surrounded by macrophage filopod ia 2 hours after the addition of specific

antibody and numerous micro-organi sms 'vere seen within phagocytic vacuoles. Some of the intracellular M . o vi pneumonia e organisms appeared normal while others appeared partially or completel�

degraded.� Twenty four hours after the add ition of specific antibody , intracellular M . ovipneumonia e organisms had been d igested.

A new procedure for col lection of alveolar macrophages was developed . The procedure provides an alternative to other methods and may be particularly useful for col lecting alveolar macrophages from the lungs of large animal species such as sheep and cattl e.

Acetone was used to dehydrate macrophages for SEM with excellent results.

In conclu sion , it was found that the addit ion of specific antibody to an M. ovi pneumoni a e - ma crophage cul ture st imulated phagocytosis of these micro-organisms. This sugges ts that if sheep gain high titres to M. ovipneumoniae, their alveolar macrophages will be able to destroy inhaled M . ovipneumoni a e organisms quickly and effectively; a possibili ty which should be tested further in vi vo .

iii

ACKNOloJLEDGEMENTS

I am grateful t o the Depar tment of Veterinary Pathology

and Pub l ic Heal th , Massey Uni ver sity, for providing the opportun i t y and facilities to undert ake this work .

^I

part icularly acknowledge with grateful thanks Dr

M. R.

Alley for hi s advice , assis tance and helpful c r i t ic ism throu ghou t the preparat ion of this thes i s .

I

woul d also l ike to thank Dr

^J.K.

Clarke for many useful discussions and supplying the

^M. ovipneumoni ae

cult ures for this s tudy .

My thanks are also ex tended t o the follo•ving people ,.,rho have been of par t icular assis tance to me:

Mr s P .M. S lack who prov ided technical advi ce in the ultramicrotome studies .

Mi ss

L. M.

Fray for her kindness and help in prepar ing TC 1 9 9 medium .

The elec tron mic roscopy

^unit

o f D . S . I . R .

Mrs F . S . Wicher t s for her generous assis tance in typing the dra f t and f inal

^copy

of this manuscript.

iv

CONTENT S

ABS TRACT

ACKNOWLEDGEHENTS CONTENTS

LIST

OF

TABLES LIST OF FIGURES

INTRODUCTION

CHAPTER ONE: General review of literature Section one : The alveolar macrophage

1 .

Introduct ion

2 . Origin of the alveolar macrophage 3 . Distribution of macrophages in

pulmonary tissue

4 . The alveolar macrophage as part of the mononuclear phagocyte system

5 . Horphology of the alveolar macrophage

Section tHo : Mycopl a sma ovipneumonia e 1 . Introduction

2 .

Distribution

3 .

Horphology

4 .

Pathogenicity Section three : Phagocytosis

1 . Introduction

2 . The mechanisms

(A) Chemo taxis ( B) Attachment (C) Engulfment (D) Destruction

of phagocytosis

and digestion Section four: Hycoplasma-macrophage interaction

Page

ii iv

V

viii ix 1 3

3 3 3

6 7

7

1 6 1 6 1 6 1 7 1 8 2 0

20 20 2 1 2 3 24 25

30

V

CHAPTER

^THO:

Ma terials and metho ds

1 . Animal s

2 . Colle c t ion of macrophages 3 . Culture media

4 . Mycoplasma o vipne umoni a e 5 . Preparat ion of antisera

6 . Exper imental design

(A) Preliminary experiments

(B) In vi tro M. ovipneumoniae inf e c t ion of ovine alveolar macrophage s

7 .

Scanning electron microscopy

8 . Transmis s ion elec tron microscopy CHAPTER THREE : Results

1 , Pre l iminar y experimen t s

2 . Morphology of normal ovine alveolar macrophages

(A)

Surface morpho logy

(B)

Ultras truc ture

3. Ob servat ions on macrophage-mycop lasma interaction

(A)

Hal f hour po st inoculation with

M. o vi pneumoniae

(B)

Half hour post inocula t ion with

M. ovipneumoniae

and an t ibody

(C)

One hour post inoculat ion \<lith

M . o vipenumoniae

(D) One hour post ino cu la t ion with

M . ovipneumonia e

and antibody

(E)

Two hou rs post inocula tion with

M . o vi pneumoniae

(F) Two hours post inocula t ion ><lith M . o vi pneumoni a e and an tibody

(G) S ix and twelve hours post inoculat ion with M . ovipneumoni a e

(H)

Twelve hours post inoculat ion ><lith

M . o vipneumonia e

and an t ibody

Page 33 33 33 35 35 35 36 36 36 37 38 39 39

4 0 4 0 46

46

46

48

48

50 52

58 6 1

vi

( I) Twentyfour hours post inoculat ion M . ovipneumoniae

(J) �'entyfour hours po s t inoculation with M. ovipneumonia e and antibody

CHAPTER FOUR: Discus sion APPENDIX

BIBLIOGRAPHY

vii

Page

64 67

.72 79 82

LIST OF TABLES

Table ^__

1 . Some early terms for the alveolar macrophage

2 . The dis t rib ution of macrophages in mammalian tissue

3 . 4 .

The mononuclear phagocyte sys tem (HPS ) The major morphological differences

between alveolar and peritoneal macrophages of germ-free rat s

5 . The· structural differences bet,.Jeen the types of alveolar macrophages of man

(Giemsa s tain)

6 .

Some agents chemotactic for macrophage s

in vitro

7.

8 .

9 . 1 0 .

Hydroly tic enzymes of macrophage lysosomes

S tudies of mycoplasma-macro phage interaction

Experimental design of ^{in vitro} alveolar macrophage-mycoplasma interaction s t udy Preliminary s tudies of macrophage

recovery from lungs.

viii

Page 5

8 9

12

l3 2 2 27

3 1

3 7

39

LI S T OF FIGURES Figure

1 . The propor t ion and size of each type of alveolar macrophage in the lavage fluid from man and hysterectomized pigs.

Page

1 4

2. The "zipper mechanism" of phagocyto sis. 25 Follm,.,ing Page 3. The intrapulmonary \vashing procedure. 33

4 .

The dis tribution of normal ovine alveolar

mac rophages on a elass coverslip . x 1 000 . 40 5 . The plasma membrane of an ovine alveolar

mac rophage. x 1 2 ,000. 40

6. High magnification of the plasma membrane of

a normal macr ophage. x

1 6 , 000. 4 0 7 .

A small group o f

5

normal alveolar macrophages.

The cells are attached to the underlying cover slip by cytoplasmic veils which spread beneath a raised dome-shaped nuclear pole.

X 6 , 000, 4 1

8.

A group of

3

or

4

normal alveolar macrophages . The largest cell is beginning to form

elongated filopodia. x

4 , 000 4 1

9 . A normal ovine alveolar macr ophage. x 8 , 1 00. 42

1 0 .

A group of normal alveolar macrophages. x

8 , 1 00 44

1 1 . H igh magnificat ion of� normal ovine alveolar

macrophage. x 1 0 , 500. 44

1 2 . A n alveolar macrophage cultured wi th

M. ovipneumoni a e for � an hour. x

3,600 . 46 1 3.

Mito tic div i s ion of an alveolar macrophage

cultured \vi th ^M. ovipneumoni a e for � an

hour x

3 , 600 . l16

1 4 . Macrophage cul tured for � an hour with M. ovipneumon.ia e and spec ific ant ibody.

X

8 , 000 . 4 6

1 5 . Macrophages cultured for 1 hour with

M . o vipneumoni a e x 7 , 000 . 4 8

16. Mito t ic divi ion of an alveolar macrophage after one h6ur of incubat ion with

M . ovipneumoni a e x 3 , 400 . 4 8

ix

Figur e

1 7 . Aft er one hour o f incubation with M . ovi pneumoniae and specific antibody alveolar mGcrophages appeared in a contrac tile position. ^x 6, 000

18. The macrophage of this preparation has sent a few fine filopodia (F) towards some

randomly distributed N. ovi pneumoniae organisms (0). ^x 5, 000.

19 . At tachment bet,veen M. ovipneumoniae organisms and the base po rtion of the plasma membrane of an alveolar macrophage

(arrow) aft er 2 hours of incubation without antibody. ^x 12,000.

20. An alveolar mac rophage cultured for 2 hours with M . ovi pneumoniae and specific

antibody. ^x 8, 000.

2 1 . High magnification of the plasma membrane

of the macrophage in figure 20. ^x 20, 000.

2 2 . Mycoplasma ovi pneumoni ae organisms cultured

for 2 hours with specific antibody. x 20, 000.

2 3 . An alveolar macrophage cultured for 2 hours Hith ^M. ovi_pne umoniae organisms and specific antibody. ^x 10, 500.

24. High magnification of the cytoplasmic

contents of the c ell in figure 2 3. x 61, 000.

25. A large phagocytic vacuole containing three M . ovipncwnoniae organisms in different stages of destruc tion is present Hithin a macrophage 2 hours aft er the addition of specific antibody. x 8 2,500.

26 . An M . ovi pneumoniae organism surrounded by finger-like projections of macrophage plasma membrane 2 hours after addition of specific antibody. x 81, 000.

2 7 .

The cytoplasmic conten t s of an alveolar macrophage cultured for 2 hours with M. ovipneumoni ae organisms and specific antibody. ^x 7, 500.

X

Follmving Page

48

50

50

52 52 52

53 53

55

55

56

Fir;ure

28. After 6 hour s of incuba tio n with

M. ovipneumoniae mycoplasma-macrophage a t tachment has developed. x 7 , 000.

29 . S triking irr egularity in th e shape of an alveolar macrophoge after 6 hour s culture with M. ovipneumon.ia e ^. x 8, 000.

30 . Attachment b e tween ^a cluster of

M. ovi pneumoniae organisms and the base of ^a macrophage after 12 hour s of

incubation in the ab sence of antibody.

x. 10,000.

31 . High magnifica tion of the cell in figure 30 shm,ring a ttachment b e t1veen the micro­

organisms and the alveolar macrophage.

X 16, 000 .

32. After 12 hours of incuba tion M. ovi pneumoniae orga nisms show ^a high degree of pleomorphism.

x . 16,000.

33. After ¹² hour s of incubation with

M . ovi pneumoni ae and specific antibody, the cytoplasm of an alveolar macrophage contains a large number of spherules comparable in size with M . ovipneumoni ae . x 6,000 . 34 . High magnification of the nuclear pole of

the cell shown in figure 33. x 16, 000.

35 . The plasma membrane of an alveolar macrophage cultured for 12 hours in the pre sence of M. ovi pneumoni ae and specific antibody.

X . 1 6, 000.

36 . An alveolar macrophage culture infec ted with M . ovipneumoniae 12 hours after the addition

of specific antibody. x 600.

37 . Alveolar macrophages cultured for 24 hours

\vi th M . o i vpneumoniae organisms. ^x 3, 400.

38 . High magnifica tion of the M . ovi pneumoni a e organisms on the cell surface of figure 3 7

X 8, 000 .

xi

Follm·ling Page

58

58

59

59

59

61

61

6 2

6 2 64

64

Figure

39. Two macrophages from a 24 hour culture with M . ovi pneumoniae only. These cells appear to be trying to join each other with cord-like extensions of variable length and thickness. x 12,000

40 . TI1ese rnacrophagcs are from a culture 24

hours after the addition of M. ovi pneumoniae .

X 4,800 .

4 1 . An alveolar macrophage cultured for 2 4

hours Hith M. ovi pneumoniae organisms.

x. 25,000 .

42 . High magnification of the cytoplasm of

a macrophage after 24 hours culture with

M . o vipneumoniae organisms. x 34,000.

43 . An alveolar macrophage cultured with M. ovi pneumonia e organisms for 24 hours.

x. 8, 1 00

44 . Alveolar macrophages, 24 hours after the addition of M. ovi pneumoni a e and specific antibody. x 600.

45. The plasma membrane of an alveolar macrophage cultured for 24 hours in the presence of M. ovi pneumonia e and specific antibody. x 16, 000 .

46 . Transmission electronmicrograph of the cytoplasmic contents of a cell cultured for 24 hours with M. ovi pneumoni a e and specific antibody. x 25,000.

47 . An alveolar macrophage cultured for 2 4 hours with M. ovi pneumoniae and specific antibody.

X 1 0, 500 .

xii

Following Page

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65

6 7

6 7

6 8

7 0

70

70

70