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The role of delayed hypersensitivity in granulomatous reactions to mycobacteria
Tubercle, Lond., (1967), 48, 109
T l t E R O L E O F D E L A Y E D t t Y P E R S E N S I T I V I T Y IN G R A N U L O M A T O U S REACTIONS TO MYCOBACTERIA:
2. Reactions to intradermal injections of intact and disintegrated organisms. By JOItN D. REID.* and J. B. MACK,KY
from The lVellhtgton ttospital. New Zealand SUMMARY A comparison has been made between the reactions produced by identical intradermal doses of intact and ultrasonically disintegrated mycobacteria (BCG) in tubercfilin sensitivemen, rabbits and guinea pigs. in men and in rabbits injections of washed whole organisms gave a biphasic response with a typical delayed hypersensitivity reaction followed by a chronic granulomatous process. Injections of ultrasonicallydisintegrated organismsresulted in considerably greater reactions tat 48 hours, with absence of chronic nodular lesions. Reactions in guinea pigs were acute and showed no clear differences. These results indicate that the presence and physical properties of intact bacteria are of major imporlance in the de~,relopment of granulomas. No evidence has been found for any special antigen as the stimulu~ for this reaction. In conjunction with previous evidence showing an association between granulomatous inflammation and hypersensitivity, it is suggested that the granuloma chiefly represents a continuing delayed reaction, modified by the physical properties of th# bacterial antigens.
R~SUM~ Les autcurs ont compar6 les r6actions produites par des doses identiques de mycobact6ries (BCG) intactes et d6sintfgr6es par les ultra-sons, inject6es par vole intradermique h des homnaes, des lapin et des cobayes sensibles/a la tuberculine. Chez les hommes et les lapins les injections d'org~nismes entiers et lav6s a donn6 une r6ponse biphasique avec une r6action typique d'hypersensibilit6 retard6e suivie par une 6volution chronique granulomateuse. Les injections d'organismes d6sint6gr6s par les ultra-sons ont 6t6 suivies de r6actions beaucoup plus importantes ~. 48 heures mais sans 16sions nodulaires chroniques. Chez les cobayes, les r6actions ont 6t6de type aigu sans diff6rence nette. Ces r6sultats indiquent que l ' & a t physique des mycobact6ries intactes est d'une tr6s grande importance dans le d6veloppement des granulomes. Aucun antig6ne particulier responsable de cette traction, n'a 6t6 mis en 6vidence. Le rapprochement avec les r6sultats pr6c6dents d6montrant une association entre l'inflammation granulomateuse et l'hypersensibilit6, sugg6re que le granulome repr6sente avant tout une r6action retard6e chronique (ou prolong6e) modifi6e par les proprift6s physiques des antig~nes bact6riens,
* Present Address: Cleveland Metropolitan General Hospital, Department of Pathology, 3395 Scranton Road: Cleveland, Ohio.
!10 RI-SUMI'N Se ha hecho una comparacion .entre las reacciones que produccn idtSnticas dosis intraddrmicas de micobacterias intactas y desintegradas con mdtodos ultras6nicos (F/CC;) en hombres tuberculine-f)o.sitivos, conejos .,,' cobayos. En el lmmbre y en conejo.,, la inyecci6rt de g,Srmenes intactos lavados di6 una respucsta bifiisica, con una reacci6n tipica d e hipersensibilidad retardada seguida por un proccso granulomatoso cr6nico. La inyecci6n de g,Srmenes desintegrac~os ultras6nicamentc di6 rcaccione.,, mayo,-es a las 48 horas, pero con ausencia de lesiones nodularc.,i cr6nicas. Las reacciones en el cobav0 eran agudas y no hubo diferencias marcadas. Estos resultados muestran que la presencia y las caractcr?sticas t'isica,, del germcn intacto son fundamentales para el desarrollo de .gntnulomas. No hubo evidcncia de que fuera un antigcno especial el que estimulara la reacci6n, l)e acuerdo con hallazgo,, anteriores ~qi.mdemuestran una saociaci6n enl.re el granuloma inllarnatorio y llt hiper.,,cn,;ibilidad, se sugiere que el granuloma represenl,a sobre todo una reaccisn continuada retardada, modificada por las propiedades fisicas de los antigenos bacterianos. Introduction
The allergic inflammatory lesions which follow injections of mvcobactcria in sensilized sul!ject~ have a chronic course and a typically granulomatous appearance with macrophages, epithclioid and giant cells: and in their development delayed hypersensilivitv has been shown to be strongly associat.ed (Reid & Mackay, 1967). However, ia},perscnsitivity reaclions, as exemplilied bx the classical tuberculin response, have a short course with maximum inlensilv at 4S-72 hours ant! are characterized microscopically by mononuclear cells and polymorphonuclcar leucocytes (Kanlan & Dienes. 1959). It" delayed hypersensitivity is causally rclaled to the granuloma, some modifying element must be acknowledged. Such a faclor could be the physical nature of the baclcrial cell, which might in the process of disintegration provide a relatively prolonged release of anti~gcn. The mononuclear cells of the delayed reaction might thus be chemolactically retained in .~itu and become transformed into the macrophage, a type of development shown by Rebuck. Bovd & Riddle (1960) for the lyrnphocyte and by Spector & Lykke (1966) t\~r the monoc.vte. If, oli the other hand, the association between allergic granuloma formation and delayed hypersensitivity is not causal, some other unidentified and distinctive hypersensitivity reaction ~eizl~.t appear to be involved, as suggested by' Kawata. Myrvik & l.eakc (1964). The absence ofgranulolnas after tuberculin skin tests raises the possibility that some specific antigcn prcsen~ in bacterial cell.',, but not in the liltered culture media used to prepare tuberculin, might be responsible for the macrophage reaction. To investigate the role of the physical state and dimensions of org:-tnisms and ,,he possible involvement ot" an unidentified bacterial antigen in the development of granuiomatous inflammation, skin reactions to intradermai injections of ultrasonically disintegrated mycobacteria were studied and compared with the biphasic granulomatous reactions known to follow injections of whole organisms (Reid & Mackay. 1967). Materials and Methods Lyophilized BeG (Glaxo Laboratories, New Zealand, Limited) was obtained as manufactured for BeG vaccination. This was reconstituted with the supplied diluent to a I in 5 solution. Organisms were then twice washed in saline, reconstituted to original volume and sterilized by heating to 100~ for one hour. By courtesy of Mr. D. Liardet (Tasman Vaccine Laboratories) other ampoules of BCG were submitted to ultrasonic disintegration in the cold, the process being continued for 20 minutes until the material was water clear and no intact organisms were visible on direct examination of smears. This material was similarly diluted to a tinal I in 5 concentration. Albino rabbits and guinea pigs were sensitized by single intramuscular i,:jections of 0.33 ml.
TAllLt:.I,--EIq l:c-I o~ U.LTRASONICDlSlNII-(IRATION(U.S.D.) ON Sizi~ OVDELAYEDAND LATESKIN REACTION:AVERAGF. SiZl;i OF REAC'I1ONSiN DIFI:EREN'I'Si't('ll-S TO INIACT AND 'DISINIt-XHtATEI)BCG Guinea pigs (10)
BCG I/5 ] 5
7 14 28
9
i11111.
"7 111111.
U.S.D. B('G 1,'5
{) l l l m . 4 111111. (9)
7 nlm. 4 rnm. ~9) . 5 ii1111. 3 ii1111.(uR) . . . . . . . . . .. . ... . . . . . . . . Numbers in brackcls refer to the number still
Rabbits (10)
Patients with tuhercuhJsis ( ! 1)
BCG 1,"5 U.S.D. BCG 1/5
5 111111.
I!
i11111.
5 111111,
4 111111. ( 7 )
6 rnm, 7 ii1111. . 6. rnm.
3 rum. 17) 3 111111.(7) I ram. (3)
B('G 1/10
: 14"5ran1. II "4 II1II1. 8"4 nlm,: 11"0 nlnl. : 10.0 nlnl.
U.S.D. BCG 1t10
22.0 11"i111. 8"4 111111.(10) 9"3 ram. (7) 7.3 ram. (7) 1.6 ran1. (3)
shov, ine. visible reactions, if less than the group total.
of live reconstituted BCG. Eleven male patients undergoing treatment of aclive p u l n m n a r y tuberculosis in the Wellington Hospital and having positive tuberculin reactions to one T U of P P D were also investigated. Tests were made by intradermal injections of 0.1 ml. of intact and disinlegrated organisms. Readings were made by transparent plastic ruler, at first daily and then on alternate days, recording the greatest diameter of reaction, as well as the presence o1" ulceration and the height ot" the lesion. Results
These are suminarized in Table 1. in guinea pigs, skin injections of" intact organisms restllted in small necrotic nodules which contained thick, yellowish-white pus and which healed in I0 to 14 days. Bacieriological examin,ilion of necrotic debris which could be expressed showed intact a/bus and other skin commensals. Histological examinarnycobacteria, as well as Staphyh~r lion al 10 days showed small abscesses with surrounding oedema and a diffuse n m n o n u c l e a r reaction. Peripherally, there were small groups of epithelioid cells with plasma cells and giant cells resembling those of classical L a n g h a n ' s type. Reactions to disintegrated organisms were not ditTercnt at 48 hours but were somewhat less at later intervals. They were also associated wilh necrosis. In rabbits, reaciions at 48 hours due to ultrasonically disintegrated organisms were twice as large as those [i)llowing the same dose of" intact organisms, and by seven days had largely disappeared; There was one exception where a large red lesion relnained for 14 days. On the other hand, the nodules following injections oi" intact organisms persisted at virtually their original diameter for a period of" Ibur weeks, although showing some regression in height. Two became ulcerated. Histological examination of nodular lesions at six weeks showed occasional acute necrotic loci with polymorphs, but chiefly a diffuse plasma cell and m a c r o p h a g e reaction with giant cells and occasional loci of epilhelioid cells, Many of the macrophages had a b u n d a n t fleshy cosinophilic cytoplasm. In 11 patients with tuberculosis, reactions to ul,~rasonically disintegrated organisrns were large and averaged 22-0 ram. at 48 hours. This response was t e m p o r a r y with rapid decrease in height. Only seven patients had measurable reactions at one week, and these had virtually disappeared by three to lbur weeks. By contrast, in these same patients and at the same time, firm raised nodules of 1 cm. in dianaeter still remained at the site of injection of whole organisms and indeed, some o f these were visible and palpable at 10 or even 12 weeks. Skin ulceration developed in six patients, in five between 10th and 16th day after injection of whole organisms, and in the remaining case on the 48th day. Biopsy of two n o n - u l c e r a t e d lesions showed at six weeks a difl'use or ill-defined follicular reaction chiefly of m a c r o p h a g e s with epithelioid cells and scattered giant cells.
1 i2
1 u I! 1:..R (" I. t.!
Discussion Tile results of these experiments differed considerably according to the species of animal (Table !). In guinea pigs, reactions to whole organisms shov,'ed a rapid development of ulceraiion, necrosis and healing. Disintegrated organisms gave reactions which were somewhat less in size, but llot dramatically different. These persisted tk~i" a longer time tt,mn reaciions to Old Tuberculin bui liar shorter periods lhan reactions to intacl organisms. Allhough a tendency to more rapid rcsoliilton was apparent, no conclusive alteralions could be ascribed to disinlegration. However, significant dilTerences in response paIlerns appeared in both rabbits and in men. Skin reactions to disinlegrated organisms were considerably greater than reactions io equal do.~es of whole orgai~isms, Thereafter, the forhler disappeared relatively rapidly (\vith a single exception which persisted as a red induraled nodule for over two weeks in one rabbit ) while ihe laller proceeded to the expected indolent granulomatous nodules. While the duration of reactions to disintegrated organisnls was greater than that to Old Tul~erculin, lasling f'rom 1-2 v,'eeks ralher lh,:ln 5-7 days, it was signiiican[lv shorter than reactions to whole organisms, xvhich ~ele scarcely anx ,,u~allcr ~.lt one lllOnlh than at one week, No evidence was found for any bacterial cell antigen having special capabililies for inducing granuiomas with the prolonged course which characierized injections of \vhole organisms. If it may be assurned thai no alteration in chemical cornpositiol~ or degradalion of anligen.,, had been induced by ultrasonic treatmerit, then the observed dilTerences indicate thal extended re\cobacterial reactions in man and rabbits are delermined by lhe physical stale of inlacl baclcria. This is consistent with the report of the produclion of skin nodules after the injection of mvcob:tcieriat cell walls (Larson and others, t963i. The particle size of i\'~reign material has been considered to be of critical imporiarlce in el/citing a foreign body rnacrophage response after injeclions of colloidal silica (Shelley & Hurlc\, I961 }, and is apparently the common denominator in the similar reactions which develop in non-.sen~,ilized animals injected with mycobacieria (Rich, 1944). E. coil pneumococci, and diphtheroids (Paget, 1942) and mycobacierial and synthetic lipids (Ungar, 1955: Ungar & Mtiggleton. 1961!. In these diverse situations, cell response would appear to be determined less by any biochemical constituei~l than by the physical properties and size of material requiring disposal. Similar consider;.~iions probably apply to the allergic granuioma. Mycobacterial celt wall conslituenis have been extensively .studied as stimuli for the macrophage reaction, but these {nclude no common chemical denominator which will cover tuberculosis, berylliosis, silicosis, fungal disease, tertiary syphilis and other granulomatous diseases. Lipopolysaccharides might be invoked in many (Jarnes, 1966t but hardly in all. The diversity of stimuli again suggests that physical properties may be the rnost important common factor. The possibility of this is conceded by Kawata, Myrxik & Leake (1964) who considered the possibility of a special form of hypersensitivity direcied against insoluble macro-molecules and lipo-protein complexes. The granulomas which occasionally ti~llow injections o f soluble tuberculin (Sandor, 1958, Hurley & Shelley, 1960) zirconium or beryllium (Shelley & Hurley 1961: Epstein, Skahen & Krasnobrod, 1963) might be similarly related to the development of particulates in the form of antigen-antibody precipitates or antigen-tissue complexes. Nevertheless, delayed hypersensitivity and particulate antigen cannot be the only factors in granuloma formation, since they also occur in infections with different histological characteristics. To approach the problem fl'om another angle, a common lack rather than a common property might be the necessary prerequisite. Granulomas characteristically lack any significant polymorphonuclear leucocyte reaction. The consequent absence of acute proteolytic breakdown of tissue would permit the evolution of hypersensitivity changes which otherwise would not be apparent. With these observations and inferences, a general working hypothesis may be advanced: that allergic granulomatous reactions chiefly depend on delayed hypersensitivity, particulate antigens, and the absence of a polymorphonuclear response.
AVIAN AND HUMAN PPD
83
For all the children having a reaction size o r 4 m m . or less to b o t h small doses of. P P D in 72 hours and who were ~hen given the large doses, the correlation o f the reaction sizes in each child is shown in Figure 3. This shows a high rate o r l o w - g r a d e reactions to P P D a:'ian and a low rate of Iow-grade
601
.~WA"; P.PO. ,='rU
J
1 ~-.........i
c ..... i .......
~
~o
1o
.........i o . . . . . . . . [2.22:_-t.:.......5-~-m. . . . . . . . . . . . . . . . . . . . . . . . Fl(~. 2 Percentage distribution of induralion 1o 5 TU of human PPD and 4 TU of' avian PPD in Brisbane school children not given BCG vaccination. reactions to P P D h u m a n to the extent that the picture using the small do~es of P P D avian and P P D h u m a n is almost duplicated. The histograms (Fig. 4) also sl~owed a repetition o f the percentage distributions to each i n d N i d u a l antigen. O f all the 1012 cl'tildren in the unvaccinated group, only 22.2 ~ gave a reaction size o r 4 ram. oi" less when tested with the s m a l l or the large doses o f the avian P P D .
T l t E R O L E O F D E L A Y E D t t Y P E R S E N S I T I V I T Y IN G R A N U L O M A T O U S REACTIONS TO MYCOBACTERIA:
2. Reactions to intradermal injections of intact and disintegrated organisms. By JOItN D. REID.* and J. B. MACK,KY
from The lVellhtgton ttospital. New Zealand SUMMARY A comparison has been made between the reactions produced by identical intradermal doses of intact and ultrasonically disintegrated mycobacteria (BCG) in tubercfilin sensitivemen, rabbits and guinea pigs. in men and in rabbits injections of washed whole organisms gave a biphasic response with a typical delayed hypersensitivity reaction followed by a chronic granulomatous process. Injections of ultrasonicallydisintegrated organismsresulted in considerably greater reactions tat 48 hours, with absence of chronic nodular lesions. Reactions in guinea pigs were acute and showed no clear differences. These results indicate that the presence and physical properties of intact bacteria are of major imporlance in the de~,relopment of granulomas. No evidence has been found for any special antigen as the stimulu~ for this reaction. In conjunction with previous evidence showing an association between granulomatous inflammation and hypersensitivity, it is suggested that the granuloma chiefly represents a continuing delayed reaction, modified by the physical properties of th# bacterial antigens.
R~SUM~ Les autcurs ont compar6 les r6actions produites par des doses identiques de mycobact6ries (BCG) intactes et d6sintfgr6es par les ultra-sons, inject6es par vole intradermique h des homnaes, des lapin et des cobayes sensibles/a la tuberculine. Chez les hommes et les lapins les injections d'org~nismes entiers et lav6s a donn6 une r6ponse biphasique avec une r6action typique d'hypersensibilit6 retard6e suivie par une 6volution chronique granulomateuse. Les injections d'organismes d6sint6gr6s par les ultra-sons ont 6t6 suivies de r6actions beaucoup plus importantes ~. 48 heures mais sans 16sions nodulaires chroniques. Chez les cobayes, les r6actions ont 6t6de type aigu sans diff6rence nette. Ces r6sultats indiquent que l ' & a t physique des mycobact6ries intactes est d'une tr6s grande importance dans le d6veloppement des granulomes. Aucun antig6ne particulier responsable de cette traction, n'a 6t6 mis en 6vidence. Le rapprochement avec les r6sultats pr6c6dents d6montrant une association entre l'inflammation granulomateuse et l'hypersensibilit6, sugg6re que le granulome repr6sente avant tout une r6action retard6e chronique (ou prolong6e) modifi6e par les proprift6s physiques des antig~nes bact6riens,
* Present Address: Cleveland Metropolitan General Hospital, Department of Pathology, 3395 Scranton Road: Cleveland, Ohio.
!10 RI-SUMI'N Se ha hecho una comparacion .entre las reacciones que produccn idtSnticas dosis intraddrmicas de micobacterias intactas y desintegradas con mdtodos ultras6nicos (F/CC;) en hombres tuberculine-f)o.sitivos, conejos .,,' cobayos. En el lmmbre y en conejo.,, la inyecci6rt de g,Srmenes intactos lavados di6 una respucsta bifiisica, con una reacci6n tipica d e hipersensibilidad retardada seguida por un proccso granulomatoso cr6nico. La inyecci6n de g,Srmenes desintegrac~os ultras6nicamentc di6 rcaccione.,, mayo,-es a las 48 horas, pero con ausencia de lesiones nodularc.,i cr6nicas. Las reacciones en el cobav0 eran agudas y no hubo diferencias marcadas. Estos resultados muestran que la presencia y las caractcr?sticas t'isica,, del germcn intacto son fundamentales para el desarrollo de .gntnulomas. No hubo evidcncia de que fuera un antigcno especial el que estimulara la reacci6n, l)e acuerdo con hallazgo,, anteriores ~qi.mdemuestran una saociaci6n enl.re el granuloma inllarnatorio y llt hiper.,,cn,;ibilidad, se sugiere que el granuloma represenl,a sobre todo una reaccisn continuada retardada, modificada por las propiedades fisicas de los antigenos bacterianos. Introduction
The allergic inflammatory lesions which follow injections of mvcobactcria in sensilized sul!ject~ have a chronic course and a typically granulomatous appearance with macrophages, epithclioid and giant cells: and in their development delayed hypersensilivitv has been shown to be strongly associat.ed (Reid & Mackay, 1967). However, ia},perscnsitivity reaclions, as exemplilied bx the classical tuberculin response, have a short course with maximum inlensilv at 4S-72 hours ant! are characterized microscopically by mononuclear cells and polymorphonuclcar leucocytes (Kanlan & Dienes. 1959). It" delayed hypersensitivity is causally rclaled to the granuloma, some modifying element must be acknowledged. Such a faclor could be the physical nature of the baclcrial cell, which might in the process of disintegration provide a relatively prolonged release of anti~gcn. The mononuclear cells of the delayed reaction might thus be chemolactically retained in .~itu and become transformed into the macrophage, a type of development shown by Rebuck. Bovd & Riddle (1960) for the lyrnphocyte and by Spector & Lykke (1966) t\~r the monoc.vte. If, oli the other hand, the association between allergic granuloma formation and delayed hypersensitivity is not causal, some other unidentified and distinctive hypersensitivity reaction ~eizl~.t appear to be involved, as suggested by' Kawata. Myrvik & l.eakc (1964). The absence ofgranulolnas after tuberculin skin tests raises the possibility that some specific antigcn prcsen~ in bacterial cell.',, but not in the liltered culture media used to prepare tuberculin, might be responsible for the macrophage reaction. To investigate the role of the physical state and dimensions of org:-tnisms and ,,he possible involvement ot" an unidentified bacterial antigen in the development of granuiomatous inflammation, skin reactions to intradermai injections of ultrasonically disintegrated mycobacteria were studied and compared with the biphasic granulomatous reactions known to follow injections of whole organisms (Reid & Mackay. 1967). Materials and Methods Lyophilized BeG (Glaxo Laboratories, New Zealand, Limited) was obtained as manufactured for BeG vaccination. This was reconstituted with the supplied diluent to a I in 5 solution. Organisms were then twice washed in saline, reconstituted to original volume and sterilized by heating to 100~ for one hour. By courtesy of Mr. D. Liardet (Tasman Vaccine Laboratories) other ampoules of BCG were submitted to ultrasonic disintegration in the cold, the process being continued for 20 minutes until the material was water clear and no intact organisms were visible on direct examination of smears. This material was similarly diluted to a tinal I in 5 concentration. Albino rabbits and guinea pigs were sensitized by single intramuscular i,:jections of 0.33 ml.
TAllLt:.I,--EIq l:c-I o~ U.LTRASONICDlSlNII-(IRATION(U.S.D.) ON Sizi~ OVDELAYEDAND LATESKIN REACTION:AVERAGF. SiZl;i OF REAC'I1ONSiN DIFI:EREN'I'Si't('ll-S TO INIACT AND 'DISINIt-XHtATEI)BCG Guinea pigs (10)
BCG I/5 ] 5
7 14 28
9
i11111.
"7 111111.
U.S.D. B('G 1,'5
{) l l l m . 4 111111. (9)
7 nlm. 4 rnm. ~9) . 5 ii1111. 3 ii1111.(uR) . . . . . . . . . .. . ... . . . . . . . . Numbers in brackcls refer to the number still
Rabbits (10)
Patients with tuhercuhJsis ( ! 1)
BCG 1,"5 U.S.D. BCG 1/5
5 111111.
I!
i11111.
5 111111,
4 111111. ( 7 )
6 rnm, 7 ii1111. . 6. rnm.
3 rum. 17) 3 111111.(7) I ram. (3)
B('G 1/10
: 14"5ran1. II "4 II1II1. 8"4 nlm,: 11"0 nlnl. : 10.0 nlnl.
U.S.D. BCG 1t10
22.0 11"i111. 8"4 111111.(10) 9"3 ram. (7) 7.3 ram. (7) 1.6 ran1. (3)
shov, ine. visible reactions, if less than the group total.
of live reconstituted BCG. Eleven male patients undergoing treatment of aclive p u l n m n a r y tuberculosis in the Wellington Hospital and having positive tuberculin reactions to one T U of P P D were also investigated. Tests were made by intradermal injections of 0.1 ml. of intact and disinlegrated organisms. Readings were made by transparent plastic ruler, at first daily and then on alternate days, recording the greatest diameter of reaction, as well as the presence o1" ulceration and the height ot" the lesion. Results
These are suminarized in Table 1. in guinea pigs, skin injections of" intact organisms restllted in small necrotic nodules which contained thick, yellowish-white pus and which healed in I0 to 14 days. Bacieriological examin,ilion of necrotic debris which could be expressed showed intact a/bus and other skin commensals. Histological examinarnycobacteria, as well as Staphyh~r lion al 10 days showed small abscesses with surrounding oedema and a diffuse n m n o n u c l e a r reaction. Peripherally, there were small groups of epithelioid cells with plasma cells and giant cells resembling those of classical L a n g h a n ' s type. Reactions to disintegrated organisms were not ditTercnt at 48 hours but were somewhat less at later intervals. They were also associated wilh necrosis. In rabbits, reaciions at 48 hours due to ultrasonically disintegrated organisms were twice as large as those [i)llowing the same dose of" intact organisms, and by seven days had largely disappeared; There was one exception where a large red lesion relnained for 14 days. On the other hand, the nodules following injections oi" intact organisms persisted at virtually their original diameter for a period of" Ibur weeks, although showing some regression in height. Two became ulcerated. Histological examination of nodular lesions at six weeks showed occasional acute necrotic loci with polymorphs, but chiefly a diffuse plasma cell and m a c r o p h a g e reaction with giant cells and occasional loci of epilhelioid cells, Many of the macrophages had a b u n d a n t fleshy cosinophilic cytoplasm. In 11 patients with tuberculosis, reactions to ul,~rasonically disintegrated organisrns were large and averaged 22-0 ram. at 48 hours. This response was t e m p o r a r y with rapid decrease in height. Only seven patients had measurable reactions at one week, and these had virtually disappeared by three to lbur weeks. By contrast, in these same patients and at the same time, firm raised nodules of 1 cm. in dianaeter still remained at the site of injection of whole organisms and indeed, some o f these were visible and palpable at 10 or even 12 weeks. Skin ulceration developed in six patients, in five between 10th and 16th day after injection of whole organisms, and in the remaining case on the 48th day. Biopsy of two n o n - u l c e r a t e d lesions showed at six weeks a difl'use or ill-defined follicular reaction chiefly of m a c r o p h a g e s with epithelioid cells and scattered giant cells.
1 i2
1 u I! 1:..R (" I. t.!
Discussion Tile results of these experiments differed considerably according to the species of animal (Table !). In guinea pigs, reactions to whole organisms shov,'ed a rapid development of ulceraiion, necrosis and healing. Disintegrated organisms gave reactions which were somewhat less in size, but llot dramatically different. These persisted tk~i" a longer time tt,mn reaciions to Old Tuberculin bui liar shorter periods lhan reactions to intacl organisms. Allhough a tendency to more rapid rcsoliilton was apparent, no conclusive alteralions could be ascribed to disinlegration. However, significant dilTerences in response paIlerns appeared in both rabbits and in men. Skin reactions to disinlegrated organisms were considerably greater than reactions io equal do.~es of whole orgai~isms, Thereafter, the forhler disappeared relatively rapidly (\vith a single exception which persisted as a red induraled nodule for over two weeks in one rabbit ) while ihe laller proceeded to the expected indolent granulomatous nodules. While the duration of reactions to disintegrated organisnls was greater than that to Old Tul~erculin, lasling f'rom 1-2 v,'eeks ralher lh,:ln 5-7 days, it was signiiican[lv shorter than reactions to whole organisms, xvhich ~ele scarcely anx ,,u~allcr ~.lt one lllOnlh than at one week, No evidence was found for any bacterial cell antigen having special capabililies for inducing granuiomas with the prolonged course which characierized injections of \vhole organisms. If it may be assurned thai no alteration in chemical cornpositiol~ or degradalion of anligen.,, had been induced by ultrasonic treatmerit, then the observed dilTerences indicate thal extended re\cobacterial reactions in man and rabbits are delermined by lhe physical stale of inlacl baclcria. This is consistent with the report of the produclion of skin nodules after the injection of mvcob:tcieriat cell walls (Larson and others, t963i. The particle size of i\'~reign material has been considered to be of critical imporiarlce in el/citing a foreign body rnacrophage response after injeclions of colloidal silica (Shelley & Hurlc\, I961 }, and is apparently the common denominator in the similar reactions which develop in non-.sen~,ilized animals injected with mycobacieria (Rich, 1944). E. coil pneumococci, and diphtheroids (Paget, 1942) and mycobacierial and synthetic lipids (Ungar, 1955: Ungar & Mtiggleton. 1961!. In these diverse situations, cell response would appear to be determined less by any biochemical constituei~l than by the physical properties and size of material requiring disposal. Similar consider;.~iions probably apply to the allergic granuioma. Mycobacterial celt wall conslituenis have been extensively .studied as stimuli for the macrophage reaction, but these {nclude no common chemical denominator which will cover tuberculosis, berylliosis, silicosis, fungal disease, tertiary syphilis and other granulomatous diseases. Lipopolysaccharides might be invoked in many (Jarnes, 1966t but hardly in all. The diversity of stimuli again suggests that physical properties may be the rnost important common factor. The possibility of this is conceded by Kawata, Myrxik & Leake (1964) who considered the possibility of a special form of hypersensitivity direcied against insoluble macro-molecules and lipo-protein complexes. The granulomas which occasionally ti~llow injections o f soluble tuberculin (Sandor, 1958, Hurley & Shelley, 1960) zirconium or beryllium (Shelley & Hurley 1961: Epstein, Skahen & Krasnobrod, 1963) might be similarly related to the development of particulates in the form of antigen-antibody precipitates or antigen-tissue complexes. Nevertheless, delayed hypersensitivity and particulate antigen cannot be the only factors in granuloma formation, since they also occur in infections with different histological characteristics. To approach the problem fl'om another angle, a common lack rather than a common property might be the necessary prerequisite. Granulomas characteristically lack any significant polymorphonuclear leucocyte reaction. The consequent absence of acute proteolytic breakdown of tissue would permit the evolution of hypersensitivity changes which otherwise would not be apparent. With these observations and inferences, a general working hypothesis may be advanced: that allergic granulomatous reactions chiefly depend on delayed hypersensitivity, particulate antigens, and the absence of a polymorphonuclear response.
AVIAN AND HUMAN PPD
83
For all the children having a reaction size o r 4 m m . or less to b o t h small doses of. P P D in 72 hours and who were ~hen given the large doses, the correlation o f the reaction sizes in each child is shown in Figure 3. This shows a high rate o r l o w - g r a d e reactions to P P D a:'ian and a low rate of Iow-grade
601
.~WA"; P.PO. ,='rU
J
1 ~-.........i
c ..... i .......
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~o
1o
.........i o . . . . . . . . [2.22:_-t.:.......5-~-m. . . . . . . . . . . . . . . . . . . . . . . . Fl(~. 2 Percentage distribution of induralion 1o 5 TU of human PPD and 4 TU of' avian PPD in Brisbane school children not given BCG vaccination. reactions to P P D h u m a n to the extent that the picture using the small do~es of P P D avian and P P D h u m a n is almost duplicated. The histograms (Fig. 4) also sl~owed a repetition o f the percentage distributions to each i n d N i d u a l antigen. O f all the 1012 cl'tildren in the unvaccinated group, only 22.2 ~ gave a reaction size o r 4 ram. oi" less when tested with the s m a l l or the large doses o f the avian P P D .