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The design of the experiment is sho\� in Tabl e 9 . Because o f the d i f f iculty in obtaining large enough numbers of

macrophag e s for sequen t ial s tudies o f phagocyto sis f rom one anima l only and the problems of processing samp les collected a t close time interval s , i t was necessa ry to d ivide this study into 2 par ts ; SEM and TEM . The source of macrophage s in both cases were healthy, Romney sheep a ged approximately one year .

For the s tudy o f the surface morphology o f norma l ovine alveolar macrophage s , a 2 mls su spens ion of ovine alveolar macrophages in TC 1 9 9 med ium wa s cul tured on coverslips in

35 x 1 0 mm plast ic t issue petrie d ishes (Falcon) and incubated

a t 3 7 oc (Table 9 ) .

Observat ions were also ma d e on infected alveolar macrophage cultur e s . Suspensions o f ovine alveolar ma crophage s in TC 1 9 9 medium mixed with a suspens ion of M . ovipneumon i a e were made .

The mixed suspension wa s e i ther cul tured alone or d iluted wi th rabb i t ant imycop lasma ant ibody and cultured on c ove r s l ip s in 35 x 1 0 mm t is sue c ulture d ishes (Falcon) at 3 7°C f o r variable t ime intervals ( Table 9 ) .

TABLE 9 : EXPERIMENTAL DESIGN OF IN VITRO ALVEOLAR MACROPHAGE - MYCOPLASMA INTERACTXON STUDY

Time interva l s

o f macrophage Norma l Macropha ges infec ted with M . ovi pneumoni a e

culture in macrophages

hours _{with ant ibody wi thou t antibody}

SEM* TEMt SEM TEM SEM

0 . 5 X X X X X 1 2 6 1 2 2 4 X X X X X X X X X

* Scanning e lec tron micro scopy (SEM) tTransmis s ion electron microscopy (TEM)

X X

X

X

X

For the s tudy of the intracellular morpho logy o f normal ovine a lveolar macrophage s a 5 mls suspension of ovine alveo lar macrophages in TC 1 9 9 med ium was cul tured in T-f la sks and

incuba�ed at 3 7 °C for 12 hour s .

Suspensions of ovine alveolar mac rophages in TC 1 9 9 were also mixed \vith a suspens ion of M. ovipneumon i a e to give 3 x 1 0 6 macrophages per ml . with and wi thou t the addi tion o f rabb it ant imycoplasma ant ibody ( Table 9). There were also cultured in plastic T-f lasks at 3 7 °C .

7 . SCANNING ELEC TRON MICRO SCOPY

On each o ccasion when s amples were taken , covers l ip s were removed f rom the cul ture and washed twice in sterile normal sal ine solut ion to remove the non-adherent macrophages

TEM X

and mycoplasams . The coverslips we re placed in modif ied Karnovsky ' s f ixat ive (see append ix I l l ) for one hour at

4°C and then washed twice with cold 0 . 1 M phospha te buf f e r ,

0

pH 7 . 2 for one hour at 4 C . Dehydrat ion o f the f ixed c overslips wa s carried ou t in a graded ace tone series at room temperature ,

for 1 0 minut e s each in 25 , 50 , 7 5 and twice in 100% , af ter 'vhich they were i�med iately air dried . Small p ieces o f cove r s lip were then cut with a diamond pencil and glued , face up , to aluminium s tubs with s ilver conduct ing paint . The preparations were coated with gol d by rout ine methods and examined under a c,..rikscan/ 100 f ield emiss ion scanning elec tron microscope .

8 . TRANSHI SSION ELECTRON HICRO SCOPY

The suspens ion of TC 1 9 9 medium containing ovine alveo lar ma crophages and M . ovipneumoni ae with or without rabbit

ant imycoplasma an t ibody wa s r emove d from plas t ic T-flasks . The f lasks were wa shed wi th 0 . 25 % tryp s in (Difco 1 : 250) in Hank ' s basic salt solution (see append ix IV) at pH 7 . 6 t o r emove the macrophages and mycoplasma s adherent to the bot tom sur face o f

the � lasks and these washings were added to the suspension .

The suspens ion wa s p laced in 1 0 m l s conical tubes and centrifuged at 1 000 g for 10 minutes . The superna tan t was drawn o f f and the cell pelle ts were f ixe d f o r two hours , at 4°C , in mod if ied Karnovsky 1 s f ixa t ive (a ppend ix I I I ) • The pellets '..re re then resu spended in 0 . 3 mls of modified Karnovsky ' s solut ion and drawn into ca pillary tubes which were centr i fuged by micro­ capillary c entr ifuge , * for 10 minu t e s . The pellets were then removed f rom the capillary tubes , washed twice with pho sphate buf f e r , f ixed second arily in 1 % o smium tetraoxid e ,washed again twice , dehydrated f o r 1 0 minutes each in 2 5 % , 50% , 7 5 % and 9 5 % alcohol and twice in 1 00% alcohol before inf il tration and embedding in plast ic capsules ( s ee appendix V) .

C HAPTER THREE

1 , PRELIMINARY EXPERIHENTS

R E S U L T S

In these s tudies an e s t ima te o f the numb er o f macrophages

which could be obta ined per '"ash '" a s made, The number and

size of the ma crophage s ob tained varied with the age , s i ze and health s tatus o f sh e e p used (Tabl e 1 0 ) . I t wa s also f ound tha t the number of a lveo l a r mac rophages obtained when s terile Hank ' s balanced sal t solut ion '..ras used was much lm..rer than that ob t a ined by u s ing normal s a l ine solut ion . Fo r this reason normal saline solu t ion w2 s used as a lavage f luid for the

remaining exp eriment s .

TABLE 1 0 : PRELIMINARY STUD IES OF NACROPH.AGE RECOVERY FROH OVINE LUNGS Sheep Amount o f fluid>'� Amount of f luid Number of

No . needed to f il l recovered f rom macro phage s the lun gs the lungs per millilitre

1 1200 mls 830 mls 8 . 700 . 000 2+ 1 400 " 900 1 1 _{1 2 . 5 00 . 000} 3 1 350 " 850 " _{9 . 200 . 000} .. .

* Normal saline solut ion

+ The lungs '..rer e inf ec ted by Dic t yocaul u s fi l ari a ( lungworm)

Thes e experiment s a l so inc luded · prel iminary scanning and t ran smi ssion elec tron microscopy s tudies of the in fec t ive

abi l ity o f· M. o vipneumonia e organisms to,..rard ovine a lveolar macrophage cultures and the ma crophage-mycoplasma intera c t ion af ter 24 hours o f incubation , The a t tachment b e tween

apparent at mul t iple point s , par ticularly at the base o f the cel l s and through sho r t and l ong filopodia .

2 . NORMAL OVINE ALVEOLAR MACROPHAGES

A . SURFACE MORPHOLOGY

Af ter 12 hours incubat ion in TC 1 9 9 med ium , ovine

alveolar macropha ges were f ound as single cells or aggregates of cells grouped toge ther and adherent to the underlying glass c over slip (Fig . 4 ) . These c e l l s were generally rounded in shape and measu red 8 to 1 7 �m in diameter . The plasma membrane wa s character ized by the pr esence of nume rous microproject ions , r idge-l ike elevations and large f lange-l ike processes (Figs . 5 and 6 ) . Mos t of the cells had sta rted to spread over the

c overslip by means of transparent veils of cytop lasm -v1hich spread beneath the nuclear pole (Fig . 7 ) . The nuclear pole was o f t en dome- shaped and covered by a relatively large numb er o f r idges and shor t ruf f les (Figs . 5 and 6 ) . The cytoplasmic veils had an undu la t ing surface with scattered ridges . Elongated f ilopodia o r pseudopodia were occas ionally visible (Fig . 8 ) and f iner p i t s 60 to 200 nm in d iame ter we re seen in mos t cel l s (Figs . 5 and 6 ) . Intracytoplasmic spherules 0 . 4 and 0 . 6 �m were also evident and may have represented lyosome s or l ipid drople t s

(Fig . 7 ) ,

B . ULTRASTRUCTURE

Normal macrophages s tudied af ter 2 4 hours varied in shape and s i ze and measured b e tween 5. 5 to 14 �m in diameter wi th a mean 9 . 6 �m . The se cells posses sed a wel l defined p lasma membrane which d i sp layed many sur face microvilli and f inger­ l ike pro j ec t ions radia t ing in various direc t ions (Fi g . 9 ) . Invagina tions o f the plasma memb rane were also common and these ext ended appreciable d istance s into the cytopl asm and

I . I I I � Figu r e 4

The distrib u t ion o f no rmal ovine alveo lar macrophages on a glass c oversl i p . The cel l s a r e d i stributed

s ingly or form small groups of 2 to 6 cells . x 1 000 .

Figu r e 5

The pla sma membrane o f an ovine alveolar macrophage . I t is charac terised by micro pro j ec t ions (m) , ridge­ l ike eleva t ions ( � ) and f lange-l ike processes ( f ) . X 1 2 , 000 .

Figure 6

High magnificat ion o f the pla sma membrane o f a normal macro phag e . No t e the surface indenta t ions ( I ) ,

Figure 7

A smal l group o f 5 normal alveolar ma crophages . The cells are a t ta ched to the underlying cover slip by cytoplasmic veils which spr ead beneath a rais ed dome­ shaped nuc lear pole . No te the undulating surface o f the cytoplasmic veils with sca ttered r idges and many spherules . x 6 , 000 .

Figu r e 8

A group of 3 or 4 norma l alveolar macrophages , The lar gest cell is beginning to form elonga ted f ilopodia . X 4 , 000 .

'

Figure 9

A normal ovine alveo lar macrophage . Thi s cell mea sured 14 �m in diame ter and possessed a well-de f ined plasma membrane wh ich displa yed many surface microvilli (V) , f inger-like proj ec t ions (F) and invagina tions ( I ) . The nucleus (N) i s roughly oval in shape and con tains a large amount o f euchromatin . The cytoplasm is exten s ive and contain s f ew mi tochondria (M) , rough (R) and smooth

could there fore be confused wi th cytoplasmic vacuoles ( Fig . 1 0 ) .

The complexi ty of the cytopla sm varied bet\ve en cells .

They con tained few o r numerous mitochond ia , s t r ip s o f rough and smoo th endoplasmic reticulum and granul es and inc lus ions o f various shapes and s izes . Although the per iphe ral cyt oplasm c on ta ined elemen t s of the endoplasmic r e ticulum; the cytoplasm of the proj ections or microv i l l i \vas devo id of membranes of the ret iculum and cons isted of f ine granular ma terial (Fig s . 9 and 1 0 ) . The in t ern al cytoplasm contained a va riety o f s tructures , mos t o f which had membranous componen t s . The membranes o f the ret iculum were characteris t ically d o t ted with r ibonuc leoprotein

( RNP) particles . The se par t icles were no t all a t t ached to the membrane ; most in fac t "VJere free wi thin the cytoplasm . They were vi s ible as dark s ta inin g granules embedded in an amorphous ma t r ix (Figs . 9 and 1 3) .

The mitochondr ia were cyl ind rical in f orm and mea sured about 0 . 3 to 0 . 4 9 �m in d iame ter and 0 . 6 to 1 . 1 �m in len g th . The number of mi tochondria seen per cell was rel a t ively small when c ompared to o ther cell types (Figs . 9 and 1 1 ) .

The Golgi appara tus \va s very extensive and occupied a large par t of the cytoplasm . I t was composed of vesicles and moderately d ense osmiophilic material \vhich formed membranous

s t ruc t ures (Fig. 9 ) .

Ves icles enclosed b y a unit memb rane "Vlere very common and · dark osmiophi l ic inclus ions with var iable d ens i t ies were n o t

uncommon (Fig . 1 1 ) .

. The nuclei \vere ova l o"r irregular in shape with one o r more inden t a t ions . Two unit memb rane s enclosed the nucleus and the se were f re quent ly in t errupted to form pores . The

•

external nuclear membrane invar iably had RNP par t icles a t t ached to i t . He terochromat in was d istributed on the nuclear margins

F igure 1 0

A group o f normal alveolar mac rophages . The plasma membrane o f these cells contains invagina tions (I) which e xtend a n appreciable distance into the cytoplasm . x 8 ) 1 0 0 .

Figure 1 1

High magn i f i cation o f a normal ovine alveolar macrophage . The cytoplasm contain s a large number

of mitochrondr ia (M) together with s trips o f smooth ( S ) and �ough (R) endoplasmic r e t ic ulum . Vesicles o f variable size are common and dark o smiophil ic inclusion s (D ) wer e occasionally seen . The nuc l e i (N) are irregular in shape with more than one invagina t ion . No te tha t the two uni t

membrane enclosing the nucleus con tains many nuclear pores ( P ) . The external nuclear membrane has RNP particles a t tached to i t . x 1 0 , 500 .

and occasionally formed clumps elsewhere in the nucleu s . The central por tion of the nucleus contained a large amoun t of euchroma t in or in te rchroma tin and nuc leoli were some t ime s ob served (Figs . 9 , 1 0 and 1 1 ) .

3 . OBSERVATIONS ON HACROPHAGE-HYCOPLASMA INTERACTION A. HALF HOUR POSTINOCULATION HITH M. OVIPNEUMONIAE

Hhen examined b y the scanning elec tron microscope , the macrophages were d i stributed mos t l y a s single cells , adherent

t o the glass covers l i p surface . The c e l l s were roughly

s pherical and occas ionally proj ec ted sho r t f il opodia (Figs . 1 2 and 1 3 ) . Mo st o f them measured be tween 8 . 4 t o 1 2 . 1 3 �m .

Ma crophage surface membranes exhib i t e d exten s ive ru f f l ing and displayed ridge-l ike profile s . Occas ional , s ingle , randomly d istributed M. ovipneumoniae organisms were seen around some macrophage s . Cel lular a t t achment be tHeen M. ovipneumoniae organisms and the plasma memb rane of macrophag e s wa s not observed at this s tage (Fig . 1 2 ) .

Smooth cord- l ike extensions , measuring 1 . 7 5 to 4 �m in thickne s s , inte rconne c t ing two macrophages wer e r e la t ively f requent f indings (Fig . 1 3 ) . Since these exten s ions appeared t o be par t of bo th c e l l s it s eemed probable tha t these

macrophages were undergoing cellular d ivisio n .

B . HALF HOUR POSTINOCULA TION HITH M . OVIPNEUMONIAE AND ANTIBODY Af ter � hour o f incubation with M . ovipneumoni a e and

rabbit ant imycoplasma an t ibody , the macrophages measured be bveen 8 to 1 1 . 7 5 �m in d iame ter and mos t c e l l s had spread extensively over the glass cove r s l i p and become f l a t t ened . They were

at tached to the covers l ip by means of transparent veils o f cytoplasm which spread beneath a raised dome-shaped nuclear pole (Fig . 1 4 ) . The nuclear pole was rela t ively smooth and

Figure 1 2

An a lveolar macrophage cul tured with M . o vi pneumon i a e

for � an hour . I t i s roughly spher ical in shape and a t tached to the sub s t ra turn by short filopodia ( f ) . The plasma membrane exhib i t s ex tens ive ru f f l ing ( r ) and displays r idge-like profiles ( i ) . There is no interac tion b e tween the macrophage and the

M. o vi pneumoniae organism ( o ) . x 3 , 600 .

Figure. 1 3

Mi to t ic d ivision o f a n alveolar macropha ge cul tured wi th M . ovi pneumoniae f o r � an hour . x 3 , 600 .

. ..,

Figure 1 4

Macrophage cul tured f o r � a n hou r wi th M . ovipneumon i a e

and spec i f ic an t ibody . The forma t ion o f extens ive cytoplasmic veils (c ) wa s a charac ter i s ti c _feature o f the cel l s i n this cul ture . Intracytoplasmic spherules ( s ) can be seen wi th in the ve il s . x 8 , 000 .

covered by fewer r id ge s than the spec imens incubated wi thout ant imycoplasma an t ibody . The cytoplasmic veils were

extensively expanded and had an undulat ing surface with

scattered ridge s . Intracytoplasmic spheru les measuring be tween 0 . 4 to 1 lJm in diamt er were evident Hithin the cytoplasmic ve il s and short filopodia sometimes extended f rom their margins

(Fig . 1 4 ) .

M . ovipneumoni a e organisms '"ere not seen att ached to the macrophage s , bu t the int racytoplasmic spheru les seen beneath the cyt oplasmic veils were compa t ible in s i z e to these micro­ organisms .

C . ONE HOUR POSTINOCULATION HITH M . OVIPNEUMONIAE

After one hou r o f incubat ion the macrophages were morpho log ically s imilar to cells f rom the � hour cul ture . HoHever , al though they Here generally rounded and measured be tween 5 . 2 to 1 2 . 9 llm in d iame ter Hith a mean of 9 llm (Fig .

1 5 ) . The cytop lasmic cord- l ike extensions be tween cells were longer , thinner and approxima tely 2 . 3 llm in thicknes s (Fi g . 1 6 ) . Mycopl asma ovipneumoni a e organ isms were d i s t r ibuted as s ingle

cells and occas ional sma ll aggre ga t ions of cells around adherent macrophages (Fig . 1 5 ) .

D . ONE HOUR POSTINOCULATION WITH M . OVIPNEUMONIAE AND ANTIBODY

Most of the macropha ges in this preparation measu red