THE PATHOLOGICAL CHANGES PRODUCED BY ANTI-LUNG SERUM

JOHN READ * From the Department of Pathology, University of Sydney , and the

Thoracic Unit, Rogal Pr ince Alfred Hmpital, S y d n e y

ZXPERLMENTAL lesions of many tissues resulting from the parenteral injection of appropriate oytotoxic antibodies were first described in the latter part of the last century.

Pcarce (1904) showed that many of the ‘‘ specific ” effects produced by such antisera were due to contamination by hwmolysins and hamagglutinins, and introduced the technique of perfusion t o free tissues from blood before using them as antigens. In the light of his results most workers concluded that antibodies against kidney substance, and against somc of the blood cells were the only cytotoxic antibodies with specific actions in vivo. It has not been possiblc to produce hetero-antibodies against tissues for which auto-antibodies are known to occup, e.g., brain tissue (Kabat et al., 1947), testicular tissue (Freund ef al., 1953, 1954) or heart muscle (Cavelti, 1947).

Nephrotoxic nephritis, produced by injection of anti-kidney antibodies into the original donor species, has been studied in great dctail. Earlier emphasis on tubular lesions was amended by Wilson and Oliver’s (1920) description of predominantly glomerular damage. Masugi (1933, 1933-34), Smadel (1936- 1937), and Seegd et al. (1955), among others, have traced the evolution of the process through acute, subacute and chronic stages morphologically similar to human glomerulonephritis. Antibodies against other tissues such as placenta (Seegal and Loeb, l946), lung, heart, and intestine (Baxter and Goodman, 1956) also produce nephrotoxic nephritis after parenteral administration. This is possibly due to their content of antibody to capillary basement membrane, for Goodmen et al. (1955) have shown that capillary basement membrane is the essential antigen evoking production of nephrotoxic antibodies.

Rabbit anti-rat-lung antibodies have beon prepared by several groups. Pressman and Eisen (1950) have shown by radioactive iodine tagging, and Mellors et al. (1955) by fluorescence techniques, that these antibodies localise selectively in both lung and kidney after intravenous administration to rats and that they persist for longer periods in the kidneys than in the lungs. No pathological changes are described in the lungs as a result of this antibody localisation, nor is there any reference to the production of such changcs in the older literature. Otto (1953-54) demonstrated that ncphrotoxic nephritis without specific pulmonary lesions often followed intravenous and subcutaneous injection of rabbit anti-rat-lung serum into rats.

This paper describes experiments in which rabbit anti-rat-lung serum was administered to rats by Tarious routes. Intratrachestl injection of such sera leads to a characteristic series of pulmonary changes.

* Present address : Department of Medicine, University of Sydney, Australia. J. PATH. BACT.-VOL. LXXVI (1968) 403

404 J . READ

MATERIALS AND METHODS

Albino rats of indeterminate local stmin and of either sex from 3 to 6 months old, and farm-bred local male rabbits weighing about 2 kg. at the beginning of the experiments, were used.

Rat's were anssthetised with ether and 5.00 units of heparin injected into the superficial femoral vein. The chest was opened and the lungs were perfrised with warm sterile 0.9 per cent,. saline through a needle inserted directly into the right ventricle until they were macroscopically blood-free. They were then aseptically removed from the chest, separated from mediast'inal structures, and cut into small pieces. Aft,er they had been frozen solid or mixed with sterile powdered glass, they wcre iinely ground in a mortar and mixed with sterile 0-9 per cent. saline to an approximately 20 per cent. suspension fine enough to pass through a 19-gauge needle. This was kept in tho refrigerator and used within a week of preparation. Random histological sections from portions of these perfused lungs showed only a very occasional erythrocyte in t.he blood-vessels. Prior intravenous injection of heparin was essential for complete clearing of red cells from t'he lung vessels.

Rabbits werc injected intraperitoneally with 20 per cent. rat-lung suspension three times a week ; one pair of rat lungs provided the material for three injections. A basic course of immnnisation consisted of 12 such injections over 4 weeks. Seven days after the last injection the first bleedings were taken from the ear veins and 75-100 ml. of blood were withdrawn during the subsequent 7-10 days. The serum was separated and stored at -20" C. Boostor courses of antigen as ncll as complete 4-week periods of re-immunisation were given later and further bleedings taken. ,411 sera were heated at 56" C. for 30 min. before use to diminish primary toxicity.

The met'hods used t o demonstrate and assay anti-rat-lung antibodies in the rabbit serum were the Ouchterlony (1948) gel- diffusion technique and a number of precipitation techniques undcr varying conditions of temperature, time, and antigen-antibody concentration.

Serial dilutions of test sera were mixed on a tile with equal volumes of a 5 per cent. suspension of rat red cells in 0.9 per cent. saline, and left at room temperature for 30 min. Agglut,ination was judged macroscopically and doubtful results checked microscopically.

One volume of washed rat-red-cells was added to 2 volumes of serum, left for 30 min. at room temperature, and removed by centrifugation. Absorbed sera were invariably slightly stained with hzmoglobin and showed no hzmagglutinating or hEmolytic properties in. witro. All specimens of serum R313 and most of R312 (vide iqfra.) were absorbed with rat red cells before use.

The centrifuged deposit from a batch of €reshly prepared rat-lung antigen was added to a small portion of serum R3/3 from which anti-rcd-cell antibodies had been absorbed. The suspension w-as thoroughly mixed and set aside for 2 hr at room temperature, after which the particulate matter was removed by further centrifugation.

A d m i n i s t r a t i o n of ant i - lung serum to rats. Anti-rat-lung sera were administered to rats intravenously, int'raperitoneally or intratracheally, under brief ether anesthesia. For intratracheal injections the technique (Magarey, 1951) consisted of passing a bliint 17-gauge needle throngh thc mont,h and larynx into the trachea of an anmthetiscd rat under direct vision. In the present series the needle was passed on gently into one or other main bronchus and 0-76 ml. of serum slowly injected.

Control experiments. Fifteen rats were given 1-7 intratracheal injections of 0-75 ml. of normal rabbit serum handled and treated in the same manner as the immune sera. Thcy were killed 2-12 days after the first injection, i.e.,

Animals.

Prepara t ion of rat- lung antigen.

P r e p a r a h n a of ant i - ra t - lung serum.

Assay of a n t i - l u n g anbibodies.

Assay of hcemagglutinating a.ntdbodies.

Absorpt ion of anti-red-cell antibodies.

Absorpt ion of an t i - lung sera zvidh rat- lung.

PNECLMONOTOXIC PNEUMOiVIA 405

1 to 7 days after the last. Eleven rats were given one intratracheal injection of normal rabbit-serum gamma-globulin and killed after similar intervals. Fourteen apparently normal rats were killed and their lungs prepared and examined in the same way as those of the experimental animals.

Anti-rat-red-cell serum (titre 1 in 256) was prepared by immunising a rabbit against washed rat-red-cells. Two rats were given a single intratrachcal injection of 0.75 ml. of this serum and 2 others a similar volume of a 1 in 5 dilution of the serum in 0.9 per cent. saline. Onc from each group was killed after 24 hr. The second rat from the first group died after ’72 hr, and the fourth rat was killed after 8 weeks.

Pathological procedures. At appropriate intervals after one or more injections of serum, rats were killed with ether. The thorax was opened and the lungs were examined and then re-inflated to their original size in situ with 4 per cent. formaldehyde through an intratracheal cannula at a pressure of a few- centi- metres of saline. The thoracic organs were removed en bloc and fixed in formalin. Blocks from the lungs and from othcr organs were cmbcdded in paraffin ; sections were cut at 5 p and stained as a routine with hzmatoxylin and oosin. Selected sections were st$ained with Laidlaw’s reticulin stain, Van Gieson’s stain and phosphotungstic acid-hwmatoxylin.

RESULTS Assay of anti-rat-lung antibodies

No in-vitro method used gave any evidence that our “immune” sera contained any anti-rat-lung antibody. A crude form of bio-assay was therefore relied upon-namely the effects produced by the sera on the lungs of living rats. Four separate batches of rabbit anti-rat-lung serum were prepared. These were R3, RB, R3/2, and R3/3, in increasing order of potency in this test. Hzmagglutinin titres ranged from 1 in 8 (R3) t o 1 in 64 (R3/2 and R3/3).

Intravenous and intraperitimeal injection of rabbit anti-rat-lung serum

Though these were given slowly from a tuberculin syringc they invariably led to attacks of acute pulmonary cedema, w-hether the serum had been absorbed with rat red cells or not. Two rats died during the intravenous injection of 1 ml. of serum, and two others within 15 and 45 min. of completion of a similar injection. One animal died of progressive hEmolytic anzmia 5 days after completion of a series of intravenous injections totalling 4.5 ml. of unabsorbed serum R3, and 2 were killed after 19 and 27 days. At autopsy the lungs of the first 5 animals showed only varying grades of pulmonary cedema, whilst those of the last 2 were normal. No lcsions were found in other organs, and a nephrectomy specimen obtained from one animal after 14 days was also normal.

Four rats were given repeated intrapcritonoal injections of anti-lung serum in total doses ranging from 3.25 to 11.5 ml. There was no clinical evidence of pulmonary cedema, and the animals survived until they were killed 3-18 weeks later. No abnormalities were found in the lungs, kidneys or other organs. A nephrectomy specimen from one animal at 10 days was also normal.

Seven rats were given intravenous injections of anti-lung serum.

Intratracheal injection of anti-lung serum After intratracheal injection of 0.75 ml. of serum rats recovered rapidly

from the effects of amsthesia. Respirations were noisy but the animals were not unduly distressed and ran about freely. After 15-30 min. their fur became elevated, ibey lay quietly in the cage and their breathing became rapid and

J. PATH. BACT.-VOL. ZXXVI (1958) 2 c 2

406 J . READ

laboured. This phase lasted from 30 min. to 8 hr and, in all animals excep6 2 that died from respiratory insufficiency after 8 hr, was followed by a return to apparent normality.

Effects of serum R3!2 and R3/3. At 8 hr (fig. 1) serum completely fills some alveoli, whilst in others it is compressed against the alveolar walls in the form of hyaline membranes. The alveolar lining cells have increased in size and number and protrude into the alveolar lumen. Cells of identical type lie free in the serum as large oval mononuclears. There is a considerable exudation of polymorpho- nuclear leucocytes, most of which lie in the substance of the alveolar walls and on their surface. Areas of focal intra-alveolar hzmorrhage are associated with capillary congestion, but there is no sign of capillary thrombosis or damage to larger vessels.

At 16 hr polyrnorplionuclear leucocytes cover and permeate the alveolar walls and form a moderate intra-alveolar exudate. Among them lie a few eosinophils and mononuclear cells, both in greater numbers than at 8 hr.

At 24 hr the first macroscopic changes appeared in the lung. The affected areas were more solid, were red to brown in colour, and did not collapse or inflate to the same degree as surrounding lung tissue. The pleural surface was normal.

Microscopically there is still serum in the alveoli, but this is over- shadowed by the cellular reaction (fig. 2). The alveolar spaces contain large masses of neutrophils and eosinophils ; many of the rieutrophils are degenerate and fragmented. Mononuclear cells are less numerous, and of 2 types-those with an oval or elongated darkly-staining nucleus and very litt.le cytoplasm, and those with a large oval vesicular nucleus and abundant cytoplasm. Some lie singly or in small clumps among the granulocytes, others form clusters partly filling an alveolus or attached to its wall, whilst still others are arranged as a partial or complete cuboidal alveolar lining epithelium.

In a few areas (fig. 3) there are peculiar granulomatous structures in the form of bulbous cellular protuberances into alveoli and alveolar ducts, attached to the alveolar wall by a thick stalk. They are com- posed of mononuclear cells and occasional spindle-cells, and their surface is covered by a layer of cuboidal epithelium continuous a t the base of the stalk with the hyperplastic alveolar lining epithelium. A few giant-cells with abundant cytoplasm and 7 or 8 nuclei are present, pesumahly arising from mononuclear cells.

In the most densely consolidated regions alveolar walls cannot be clearly delimited. In less densely affected areas they are frequently widened by capillary dilatation, interstitial mdema, and mononuclear- cell infiltration. Though the walls and lumina of pulmonary arteries and veins show no distinctive changes, the perivascular spaces are widely dilated as if by cedema fluid and contain varying proportions of neutrophils, eosinophils, large mononuclears and scanty lympho- cytes. Small bronchi leading to unaffected areas of lung are normal,

PLATE LXXXV

I h c ? . 1 .--8 hr. Polymorphs clustcrod about BTG. 2.-24 hr. Exudate of ncutrophils, t&lvoolur septa. H rcrnatoxylin and eosin. eosinophils and mononuclears. H. and E. x 300. X 300.

Bra. 3.--24 hr. Grrtnulomatons bud-like lesions. H. and N. x 300.

PIG. 4.-48 hr. Nxudate of mononuclear cells with vesicular nuclei. .K. end E. X 300.

FIG. 5.-48 hr. Lwge intra-alveolar giant-cell. FIG. 6.-'72 hr. Pattern of interstitid H. and E. X 300. thickening. H. and E. X 76.

Sections of rat lung at intervals after intratrmheel injection of anti-rat-lung serum.

407

lout those associated with inflamed areas show mucosal lesions- hyperplasia of the lining cells of the mucosa, which is sometimes several cells thick, and numerous goblet cells. The remainder of the 1)ronchial wall is normal and peribronchial lymphocytic cuEng is usually absent. The bronchial changes are well marked up to 72 hr after injection, after which they rapidly regress.

At 48 hr, though there was little macroscopic development in the lungs, there is considerable histological alteration ; the cellular exudate has become predominantly mononuclear (fig. 4). Neutrophils are absent in many areas and those that remain are largely degenerate. Eosinophils on the other hand are well preserved and sometimes form compact masses among the mononuclears, which, though more numerous, show the same distribution as at 24 hr. In the granulomatous buds the cells have become more ovoid and are arranged with their long axes parallel to the covering epithelium, so that the whole nodule takes on a whorled appearance. Alveolar-wall thickening is now due almost entirely to cellular infiltration ; capillary dilatation and inter- stitial cedema have largely subsided. Giant-cells have increased in size and number. They are round, oval, or less often irregular in shape, and contain 5-20 nuclei in the plane of sectioii. They contain no inclusions or obvious debris and correspond to the Langhans type of giant-cell. Some of them reach an enormous size (fig. 5). They occur free within alveolar spaces, attached to alveolar walls or in the bulbous septa1 protrusions already described.

As the granulocytes disappear from the alveoli they increasc in number in the widely dilated perivascular spaces, which are packed with neutrophils and eosinophils, a few large mononuclears and an occasional lymphocyte. Reticulin staining demonstrates that the connective-tissue framework of the lung is intact and that there is no significant atelectasis.

At 72 hr the lungs of most animals in this group contained smooth solid pale-red areas. Those of the remainder, apart from being firmer than usual, were macroscopically normal. Both groups show identical histological changes.

Two types of lesion are present. Where consolidation is fairly complete few granulocytes remain, arid the whole lung substance is heavily infiltrated with large mononuclear cells. These cells stain more deeply and are more discrete and elongated than previously. Giant-cells are numerous. More common than these solid areas are regions (fig. 6) where a clear distinction can be made between alveolar cont,ents and alveolar walls and where the pattern of lung st,ructure stands out in exaggerated relief with an almost glandular appearance. The alveolar walls (fig. 7 ) are thickened by mononuclear-cell infiltra- tion and covered by a complete lining of more attenuated cells. Alveolar-wall capillaries are dilated in some regions, bloodless in others. As well as variable amounts of hzmorrhage the alveolar spaces contain large or small clusters of pulmonary macrophages. Dilated

408 J . READ

perivascular spaces are packed with approximately equal numbers of eosinophils and large mononuclears.

Silver staining demonstrates that most alveolar walls contain normal reticixlin fibrils. In one animal, however, there is focal frag- mentatmion of the reticulin, involving short segments of alveolar walls or a t most a whole partition between adjacent alveoli. Xo corres- ponding abnormalities can be deteetcd in sections stained with hzmatoxylin and eosin or with phosphotungstic acid-hzematoxylin.

At 96 hr the lungs were either macroscopically normal or contained grey to pink solid areas slightly lobulated on the plcural surface.

Corresponding to these gross appearances are predominantly solid and intcrstitial histolo$cal lesions. Fibroblasts have bccomc prominent among t h e mononuclear-cell infiltration ; in the solid areas they follow the al\ eolar wall pattern, whilst the alveoli are stuffed with histiocytcs. I n the areas of looser testare (fig. 8) alveolar exudate is reduced to a few macrophages, and the alveolar walls contain variable proportions of ovoid mononuclears and fibrohlasts. The alveolar lining epithelium is much less prominent, but still clearly delimits thc boundaries of the septa. Giant-cells of the Langhans type are very numerous (fig. '3) ; sonie loq--power fields cont'ain up t o 12 such cells. Silver staining shows a little hyperplasia of the alveolar-wall reticulin, but minimal intra-alveolar organisation.

The lungs of one animal showed scattcred round or oval areas of tissue necrosis varying from i -2 high-power fields in diameter (fig. 10). These areas are composed of a mass of lumpy eosinophilic material in which it is still possible to trace thick structureless alveolar walls and round necrotic cell bodies ; among these lie many fragments of nuclear debris. At the edges there is a sharp transition to the general pulmonary consolidation, and here fibroblasts and ovoid mononuclears show some palisading. Silver staining of these lesions (fig. 11) dcmonstrates ;i transition from areas with norinal ret(icu1in in the alveolar walls, through a zone where cells seem normal but the reticulin is fragmented, to the necrotic centre where the cells are dead and reticulin fragmenta- tion is extreme. The reticixlin changes in the intermediate zone correspond with those described in focal areas after 7 2 hr.

An indisputable arteritis was not seen in any animal, but in one rat killed after 48 hr arid in two killed after 96 hr suggestive lesions were observed. Collections of polymorplionuclear leucocytes appear to infiltrate short segments of the wall of small muscular arteries throughout the lung. This cellular infiltrate is quite distinct from that in the perivascular spaces. No fibrinoid necrosis or other form of degeneration of the vessel wall itself can be demonstrated, nor are there any endothelial changes or thrombosis. The necrotic lesions mentioned above do not occur in the same lungs as the vascular changes.

After 14 days the lungs mere either macroscopically normal or else yellow-white and somewhat small. Both varieties show the same histological changes (fig. 12).

.I. PATH. IIACII!.. ---vor,. nxyr

PNEUMONOTOXIC PNEUMONIA

PLATE LXXXVI:

1Crc:. 7. - .72 hr. Mononuch!ar-c:ell thickening FIG. 8.-96 hr. Fibroblasts in the tclveolw of alveoler walls. H . and le. x 300. sopta. H. and 1C. x 500.

VTG. IO.---96 hr. M:MS of necrcwcd tissue. Palisading at margin. H. and E. X 300.

VLG. 1 1 .-96 hr. ‘I’issno nocrosis. hidlaw’s FIG. 12.-14 days. Early interstitial fibrosis. rc?tic:ulin stain. X 300. H. and K. X 150.

Sections of rat lung at intervals eflm intratracheal injection of anti-rat-lung scrum.

. I . PA!llH. I%ACT.--.Voi.. :I,XXVJ

PNIWMONOTOXIC PNJEIJMO‘NIA

PLATE LXXXVII

L~T(:. 13.-14 days. Increase oi roticulin in Flu. 14.-21 days. Loose fibrosis of alveolar ablvoohr walls. hidlaw’s s t t h i n . X 150. sccpta. H. and $1. X300.

I!’CC. 15.-21 days. More compact fibrosis of sopta. R. and N. x 300.

E’TG. 16.--81 days. Incroasing amount of re t id in . Laidlaw’s stain. X 300.

WIG. 17.-72 hr after serum FIQ. 18.-10 days after serum R6. Thick WIG. 19.-24hraft~rsenun 1x5. Woll doveloped bud- bands of fibroblastic tissue. H. arid E. R3. Fibrinous bud-like liko lesion. H. and E. x 150. lesion. H. and E. X 300. x 300.

Sections of rat lung at intervals after intratracheal injection of anti-rat-lung serum.

PA’E UMONOTOXIC PNB UMONIA 409

The alveolar walls are considerably thickened, both uniformly and in localised nodules, by infiltration with fibroblasts and an increased amount of connective tissue. A few medium-sized mononuclears remain in the alveolar walls and spaces. In many areas there is no obvious alveolar epithelium, other alveoli are lined by a few flattened cells, and a few retain a partial lining of cuboidal cells. Alveolar-wall capillaries are almost completely absent. The alveoli are reduced in size and their lumen is encroached upon by the proliferative process in the alveolar waUs. T h e n atelectasis is superadded all trace of alveolar structure is lost and conglomerate scars are produced. lntra- alveolar organisation, though not completely absent, is much less important than the interstitial process.

Silver staining (fig. 13) demonstrates a well-marked increase in the reticulin content of the lung. Alveolar walls contain many fibrils of reticulin, most of which follow a twisted course along the axes uf the septa ; but there arc also numerous tiny side-fibrils. Strands of reticulin invade occasional alveoli, but the bulk of the process is interstitial. Even in the conglomerate scars the heaviest reticuliii deposition is in the original alveolar septa. Van Gieson staining demonstrates only small amounts of collagen among the reticuliii fibres of the septa.

Slightly less than half the animals killed later than 14 days after injection had lungs macroscopically and microscopically normal, or showing only the lesions of chronic miwine pneumonia. The remainder, either macroscopically normal or with small contracted areas on their surfaces, contained developing lesions.

Microscopically these lesions are more focal and disseminated than in the earlier phases, and it is not uncommon to find groups of 8-16 or even fewer alveoli showing the characteristic changes. Pro- gression (figs. 14 and 15) entails decreasing celliilarity and increasing fibrosis of the alveolar walls. Few mononuclears persist, and small numbers of fibroblasts form the bulk of the residual cells. Discret<e wavy connective-tissue fibres are more obvious, sometimes loosely arranged (fig. 14), at others more dense and compact (fig. 15). Capillaries are absent in these septa. The alveolar spaces are reduced in size and focally alveoli are completely obliterated. All trace of the origiiial intra-alveolar exudate and alveolar lining epithelium has disappeared.

Rarely, fibrosis completely fails to respect alveolar anatomy, and a band of scar tissue of the same histological age as the interstitial lesions traverses the lung substance. There is at least one shrunken fibrotic bud-lesion, but sequel= of vascular and perivascular lesions are absent.

Silver staining (fig. 16) reveals even greater increase of reticulin fibrils than that seen after 14 days. Collagen, confirmed by Van Gieson staining, forms an increasing proportion of the septa1 connective tissue in the period from 2 to A weeks after intratrxcheal injection of serum. The most advanced lesions studied (at 6 weeks) consist of

410 J . READ

dense, compact, collagenous scarring of the alveolar septa, almost identical with the appearances in fig. 15.

Effects of sera R5 and R3. These resembled the lesions produced by sera 8312 and R3/3 in all essential features, but there were some small differences, Complex cellular buds protruding into alveoli and alveolar ducts were more numerous and better developed with serum It5 than with the previous sera. One illustrated in fig. 17 shows well the cell-content of mononixclears and spindle-cells, the stalk of attach- ment and the clearly defined covering cuboidal epithelium. Such structures axe ohviously different from masses of mononuclears fortuitously lying in apposition to an al-veolar septum. A second variety of bud-like process seen especially in alveolar ducts was composed of fibrin containing a few polyrnorphonillclears and macro- phages, and covered by an attenuated layer of epithelial cells. The alveolar septa 4-5 days after intratracheal injection of serum R5 showed more widespread and abundant conversion into bands of fibroblastic tissue than warns apparent with sera R312 and R3/3, and this feature was still marked after 10 days (fig. 18). On the other hand 5erimi R85 produced an alveolar exudate containing fewer eosinophils, less perivascular-space dilatation, arid no tissue necrosis or arterial lesions.

l n the lungs of animals injected with serum R3, the first and the weakest serum prepared, graniilocytes were more numerous and persisted longer in the acute lesions. whilst the succeeding alveolar- wall fibrosis was more focal. During the acute exudative stage bud- like lesions composed predominantly of fibrin instead of mononuclear cells were seen (fig. 19). These contained scanty mononuclears and iieutrophils and were devoid of a cellular covering.

Frequency und extent of pulmonary lesions. The lesions were apparently confined to areas of lung reached by immune serum. Acute and subacute lesions usually involved the greater part of one or more pulmonary lobes while chronic lesions were far more restricted. At most there might be confluent involvement of one-third of a lobe, whilst in some animals changes were restricted to small groups of alveoli scattered throughout a lobe. Chronic lesions were never of such an extent as to render a rat obviously dyspnceic. The frequency of various changes is shown in tables I and 11.

No gross or microscopic lesions were found in the heart, liver, or kidneys. The spleen showed consistent changes after the injection of both anti-lung serum and normal rabbit serum, and there were no quantitative or qualitative differences between the 2 groups. Macroscopic enlargement was apparent after 24 hr, increased to a innximum at 5 days, then slowly subsided over the next 16 days. Microscopically there was progressive enlargement of the Malpighian corpuscles. At 5 days they consisted of masses of large pale cells surrounding the original follicles and poorly defined from the surrounding pulp. They slow17 regressed and were still

Changes i i a other orgcrns.

41 1

56 11

19 .6 ~-

larger thm normal 3 weeks after the injection of serum, but had returned to their origina.1 state 5 weeks aft,er injection.

After intrat,raclieal injection of anti-lung serum, but not of iiorrna,l rabbit serum, the superior mediastmind and cervical lymph-glands followed a cycle similar to that of the splenic corpuscles. Macroscopic

TABLE 1 Incidence of experi.mentcz1 changes after intrntracheal injection

of anti-rat-lung serum _______

So . of rats showing pneunlonotosic leainns!No. killed, at t,irne aft,er intratrscheal injection of anti-rac-lung serum (days)

1-4 ~ 5-11 12-20 1 21-26 ~ 27-35 .,-.____

~

I

I Serum

I-- ~

H 4 3: 4 ~ 5! 6 0;1 ... ! ...

i R3:3 1 7 / 1 i , ... , 2j2 1 2 4 I 1p ~ 213 1 ... ... i .._ i :::

6i 7 ~ 01 1 ' ... l i2 j ... I ... 91 9 I 2 : 3

-_-__I. _______~ ~

Total 35/37 I 7 / 1 0 I 2!3 3/6 ' 1;'3 !

enlargement was noted within 48 hr, increased up to the fifth day and then regressed rapidly. When pulmonary changes were unilateral lymph-gland enlargement was usually restricted to, or more marked on, the corresponding side of the mediastinum. The cervical lympli- glands deep to the sternomastoid muscle were sometimes enormously

TABLE I1 Lobar distribution of lesions after intratracheal injection

of anti-rat-lzccng Serum ~

I Left l ing

Wnmher of lobes examined

toxic pneumonia Per cent. lobes involved

intcr- Right Right

mediate lou-er lobe lobe i

~

Right middle lobe

58 14

24.1

For this distribution x2 = 32.3 ; n = 4 ; P<O.O1 ; i .e. the lobar distribution is highly unlikely to be a chance one.

enlarged, a group of glands from one animal forming a mass 11 x 3 x 3 mm. Histologically the glands showed non-specific hyperplasia of aU. elements ; in a few, clusters of eosinophils were seen in the afferent sinus.

Control procedures Intratracheal injection of normal rabbit serum. The immediate reaction to

the injection of normal serum W ~ S identical with that following the hiechion of anti-lung serum. After 15-30 min. the fur became elevated and the animals quiet, but there was no respiratory distress.

Macroscopically, lungs from this group were normal apart from obvious murine pneumonia. Histologically the lungs of 6 animals were complete~y

412 J . READ

normal. Two others showed a little free serum in the alveoli with no celluh rcaction. In the remaining 7 animals there were minor degrees of cellular reaction ranging from a little prominence of tho alveolar lining cells (2 rats), to the exudation of trifling (2 rats) to moderato numbers ( 3 rats) of polymorpho- nuclem and macrophages. In none did the soverity of tho reaction approach that due to injection of anti-rat-lung serum and in all there were many alveoli containing e.erum but no cellular exudate.

All 7 rats showing any reaction had been killcd leas than 72 hr after injection. The changes produced by intratracheal injection of normal rabbit-sorum gamma- globulin (11 rats) were entirely similar to those produced by normal rabbit serum.

Twenty-four hours after injection of serum of titre 1 in 256 there was an alveolar exudate of polymorpho- nilclears and mononuclears, less marked than that produced by anti-lung serum. Three days after injection the nature of this exudate had hardly changed at dl and there was still abundant cell-free intra-alveolar serum. After injection of more dilute (titre 1 in 50) anti-rat-red-cell serum no abnormalities wcre demon- strated in the lungs after 24 hr and 8 weeks.

TAB^ III Lobar dist&n&m of major lesiom of chrowk murine pneumonia

Intrajmcheal hjectwn of anti-rat-red-cell smm.

- __. -_

inter- Eight

lobe l ’ ln~ mediate lower lobe

Right ! Eizlit Right middle lobe upper lobe

Spontaneous pulrmwry disease ir, the local rat colony The albino rat is subject to all grades of spontaneous chronic pulmonary

disease, ofton rcferred to a8 ‘‘ bronchiectasis ”. Innee et d. (1966) have given a full description of the pathological features of thie chronic murine pneumonia, and their findings have been largely confirmed in the local rat colony.

numbero of lobes examined . 115

Number showing lesiona . 26 I I---- I -~ -

114 I 100 92

19 93 27 i 21 I 25

a Per cent. lobes involved 29 0 1 18.4 1 25-0 1 10.7 _-

226 I _.

P Z E U M O N O T O X I C P X E UMOI1IA 413

Macroscopic lesions seen were tiny lymphoid follicles scattered over the surfaces of the lungs, abscesses of all sizes, atelectasis involving part or all of a lobe, dilatation of bronchi, and enlargement of mediastinal lymph-glands when frank abscess formation had occurred. Microscopically atelectasis varied from complete airless collapse to an appearance best described as under- inflation. In both cases varying amounts of infection were present, and there was some increase of mononuclears in the septa, which were always highly vascular. In the severer grades a little interstitial fibrosis was not uncommon at the edges of the lesion. Lymphocytic permcation of the peribronchial and perivascular spaces and of surrounding collapscd lung was a universal feature of specimens of chronic murine pnoumonia. Giant-cells were absent.

The incidence of spontaneous pulmonary lesions is shown in table 111.

DISCUSSION The techniques of the current experiments closely follow those

used for the production of nephrotoxic sera, and are very similar to those used by Pressman and Eisen (1950), by Otto (1953-54) and by Mellors et al. (1956) for the production of antibodies against lung tissue.

That it proved impossible to demonstrate the presence of anti- lung antibodies by in-vitro precipitation procedures is less disconcerting than might a t first sight appear. Those who claim to have measured the titre of anti-kidney antibodies in vitro have used various modifica- tions of precipitation techniques, and a striking feature is the range of titres demonstrated by different workers in sera which, to judge from in-vivo tests, contain about equal concentrations of antibody. These titres have ranged from 1 in 4 (Simonsen, 1953) to 1 in 64 (Kay, 1940), even to 1 in 1024 (Pressman and Keighley, 1948). Smadel (1936) found no correlation between in-vitro results and the cytotoxic action of nephrotoxic sera in viwo, and Pearce (1904) found in-vitro reactions so confusing and contradictory that he discarded then1 in favour of testing the action of sera directly in vivo. Some other writers (Heymann and Lund, 1961) have openly discarded in-vitro determin- ations, and others (Seegal et aE., 1955) make no mention of them at all.

Features supporting the contention that the changes in the lungs were produced by anti-lung antibodies are :-( 1) their appearance after intratracheal injection of serum from rabbits given repeated injections of rat-lung suspension, but not after injection of serum from normal rabbits ; ( 2 ) their occurrence even after hamagglutinating and hzmolytic antibodies had been removed from the serum ; (3) the small experiment in which changes were prevented by prior absorption of the serum with rat-lung suspensions ; and (4) the nature of the pathological changes themselves.

A mere foreign-body reaction t o the presence of serum in the lungs is excluded by the absence of such changes following int,ratracheal injection of normal rabbit serum, and these results have been confirmed in another context by Hadders and Dirken (1955). Similarly, though sera were not tested for sterility, the absence of reaction after normal- serum injections compared with the constant pattern with different

414 J . READ

batches of antiserum seems to rule out an infective factor. The entirely different lobar distribution of the experimental changes compared with those of chronic murine pneumonia (tables I1 and 111) seems to exclude any connexion between the two processes.

If it be granted that the pathological changes were due to an antibody or antibodies in the iiijected serum, what is the nature of this antibody 1 It was not the hzemolytic or the hsmagglutiiiating antibody, as is shown by the absorption experiments and by the failure of direct injection of lizemolpins to produce comparable changes in the lung. It was not due to non-specific anti-rat-connecti~~e-tissue antibody. The peritoneal cavities of rats given intraperitoneal injec- tions of antiserum were in general quite normal, and anti-lung serum injected intraderinally or subcutaneously disappeared rapidly leaving no trace and causing no delayed reaction. The absence of renal changes following large intravenous and intraperitoneal iiijections of anti-lung sera rules out the presence of significant amounts of anti- glomerular-basement-membrane antibody, which causes nephrotoxic nephritis (Goodman et al., 1956). Glomerular basement niembraiie is antigenically identical with capillary basement membrane from the lung (Goodman et al., 1955), so it maj- be concluded tha t the current specimens of anti-lung serum contained no significant anti-capillary antibodies. Antibodies against the tissues of larger vessels arid bronchi seem t o be excluded by the rarity and 1ack of severity of lesions of those structures.

Thus by exclusion we conclude that the effective antibodies are directed against the cells of the lung substance itself, namely the alveolar lining cells. This postulate will be taken up in discussing the pathogenesis of the lesions.

Masugi (1933, 1933-34) regardod the nephritis which follows the intravenous injection of anti-kidney serum as due to a simple process of reverse anaphylaxis ; kidney (antigen) plus anti-kidney serum (antibody) gives rise to antigen-antibody union with resultant tissue damage. Kay postnlatcd union of antibody with renal structures, development elsewhere in tho body of antibodies against the injected antiserum, and tissue damage as a result of reaction betm-een tho host's antibodies and the foreign anti-kidney serum attached to renal tissue. Simonsen produced evidence in favour of union between injected antibody and glomrrular tissues, with local cellular proliferation and local antibody production against tho foreign antibody. He postulated direct tissue damage if largo amounts of foreign anti-kidney antibody were injected, a inononidear proliferativc reaction if the insult were less. His thesis is in keeping with the work of Ehrich et al. (1949) who regarded the proliferative mononuclear reaction seen in various tissues after intravenous injection of foreign protein as a function of antibody formation.

I t is likely that a mechanism such as that postulated by Sinionsen must be invoked to explain the genesis of the lesions in the present experiments, which difl'er in many ways from those of a simple arthus reaction occurring within the lung. The two most careful studies of the intrapulmonary Arthus reaction (Fried, 1933 ; Cannon et d.,

1941) are representative in demonstrating that the primary lesion is one of gross damage to large and small vessels in the lung. Vascular lesions in the present series were few and unconvincing.

Simonsen’s paper should be consulted for full details of his work and hypothesis on nephrotoxic nephritis. Suffice it to say in this context that a translation of his views to the present problem would postulate the following course of events. A severe insult by antibodies localising in the lung would cause frank tissue necrosis. If lower concentrations of antibody were used t,here would be union between antibodies and the target cells (alveolar lining cells) with result’ant proliferation of these ceVs and local antibody formation. The pathological observa- tions made are in accord with this hypothesis if we regard the mono- nuclear cell proliferation as an expression of alveolar-lining-cell multiplication. The more complex structures-giant-cells and bud- like lesions-might be interpreted as further manifestations of alveolar- lining-cell proliferation, and indeed lesions such as that depicted in fig. 17 bear a striking resemblance to t3he so-called allergic granuloma of Goddard J1947).

Pulmonary lesions were almost constantly present in the first 4 days after intratracheal injection of antiserum (table I). I n the 2 animals that failed to show such lesions serum may have failed to reach the lungs for technical reasons. This frequency suggests that the lower incidence of abnormalities in animals killed after longer intervals was not due to failure of development of such lesions, but rather due to resolution of pre-existing lesions. This is supported by the finding that chronic changes were never as extensive as the acute and subacute ones. It is postulated that a pulmonary reaction invariably developed after successful intratracheal injection of anti- lung serum, and that it followed one of two possible courses-resolution or interstitial fibrosis.

The lobar distribution of the lesions (table 11) supports the contention that they developed only in relation to injected antiserum. The tracheal cannula passed more frequently into the left main bronchus than into the right, and the animals were held vertically aa serum was injected. It will be seen that left-sided lesions were more frequent than right-sided ones, and that the right intermediate and lower lobes were affected more often than the right middle and upper lobes. Chronic murine pneumonia on the other hand affected all lobes approximately equally (table 111).

No obvious pulmonary change was produced by intrayenoris or intraperitoneal injection of anti-lung serum. I n other words these experiments are not completely analogous to those on nephrotoxic nephritis. Pressman and Keighley injected the gamma-globulin fraction of an anti-lung serum labelled with radioactive iodine intra- venously and demonstrated considerable localisation of radioactivity within the lung, as well as in the kidney. This may not be strictly germane to the present problem. Such localisation of radioactivity

416 J . READ

may have been due to the anti-capillary-basement-membrane fraction which is often present in such antisera, though it is apparently absent in mine. It is conceivable that the anti-alveolar-lining-cell factor which has been postulated as the cause of the present reaction is unable to reach its target cells from the blood-stream in acute experi- ments. It might be noted that nephrotoxic nephritis depends upon antibodies against glornerular basement membrane which is in direct contact with circulating blood and antibodies. In the present circum- stances it is suggested that the target structures are adequately separated from the blood by those structures of the alveolar wall lying between capillary and alveolar lining cells.

By analogy with nephrotosic nephritis, the effect of anti-lung sera on the lung might be termed pneumonotoxic pneumonia.

The possible clinical inferences which might be drawn from these experimental findings wil l be discussed elsewhere (Read, 1955). It, will be suggested that the morphological features produced may have their counterpart in human lungs derived from some cases of rheumatic and rheumatoid disease, polyarteritis nodosa, lupus erythe- matosus, acute glomerulonephritis, Wegener's granulomatosis and scleroderma, as well as the syndrome of diffuse interstitial pulmonary fibrosis described by Hamman and Rich (1944).

SUMXARY

Intratracheal, but not intravenous or intraperitoneal, administra- tion to rats of rabbit anti-rat-lung serum leads to a distinctive series of changes within the lung (pneumonotoxic pneumonia). There is early hemorrhage and exudation of neutrophils and eosinophils, followed by active mononuclear-cell proliferation. The latter leads t o the formation of clusters of alveolar inacrophages, partial or complete alveolar epithelial linings, cellular thickenings of the a.lveo1ar septa, and specialised granulomatous buds protruding from alveolar walls, as well as giant-cells of Langhans type. Tissue necrosis is rare, and evidence of arteritis uncommon and unconvincing. Focal capillary damage is absent.

The *lesions may proceed to complete resolution or t o fibrosis of the alveolar septa, with occasional focal collapse and intra-alveolar organisation.

The pathogenesis of the changes has been discussed in terms of an allergic basis on the iiephrotoxic nephritis model.

Thanks are dne to Professor F. R. Magarey for his interest, encoixragement, and help. This work was carried out during the tenure of a Royal Prince Alfred Hospital Thoracic Unit Research Fellowship. The photographs were prepared in the Department of Illustration, University of Sydney.

PLNEUMONOTOXIC PNEUMONIA

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J. PATH. BA0T.-VOL. LXXVI (1958)