ON THE DISSOCIATION OF 1MMUNE-BODY FROM ERYTHROCYTES WlTHIN THE LIVING ORGANISM.‘

By ROBERT MUIR, M.D., F.R.S., ProfcssoT of Pathology in the University of Clnsgow, and J. W. M‘NEE, M.B., Ch.B., MQunn Scholar in PLtthology.

E’ram lice I’athological Laboratories of GZaagow University a d Glmgmn Western InJErmnry.

IX our recent payer (1 9 1 2 ’) on the ‘‘ L4n~niia produced by a Hcniolytic Serum ” we showed that the amount of blood destruction produced by the injection of the immune-body was much greater than what would correspond to the amount of lysis in test-tube experiments. I n con- sidering this result the time element was taken into account, but we found no evidence that a corresponding length of time given for the ocmirrence of lysis in vitro would explain the great difference observed. The possibility of dissociation of immune-body after lysis of the cor- puscles in the circulation was then considered, and experiments bearing on this question were undertaken. The results of these are not put forward as explaining entirely the great difference between lysis in vitro and the amount of blood destruction in the living body, but they are of iinyortance as actually showing that a considerable degree of blood destruction can be brought about when the iinniune-body is introduced in combination with red corpuscles. They also indicate that tho immune-Lody may be separated from the receptors of the red corpuscles to a much greater extent in the living body Lhan in vitro ; in the latter ca8e the separation depends upon physical dissociation, but in the former, active separation may possibly be effected by the living cells, and immune-body thus set free to a greater extent. In our present series of exyeriincnts the amount of immune-body com- bined with corpuscles injected was nevcr more than a hcmolytic dose, i.e. the minimum amount suEcient to produce complete lysis in the presence of sufficient complement. The effects following the injection were accordingly due to immune-body which had been concerned in the original destruction of the corpuscles introduced.

[Received Jnue 29,1912. J Towards the expenses of this researcli n grant was obtained from the Carnegie Trustees, for which re have pleasure in recording our indebtedness.

DISSOCIA TION OF IMMUNE-BODY 93

It has now been demonstrated in a large number of instances that the union of antigen with anti-substance can to a certain extent be split up and the two components obtained separate.

In the case of hmmolytic sera this was first shown by Mogenroth (1903 2)

and by Muir (1903 3) independently, who found that when to a suspension of red corpuscles combined with several doses of immune-body fresh corpuscles were added, a sufficient amount of immune-body passed over to the latter to produce complete lysis of them when complement was added. Previous to this, v. Dungern (19004) had found that when red corpuscles saturated with immune-body were injected into an animal they did not lead to the develop- ment of immune-body, a result which was apparently in conformity with Ehrlich’s view that the formation of immune-body depended on the combin- ing group of the antigen being free or unoccupied. A little later, however, Sachs (19016), in repeating these experiments, found that in a certain nunibcr of cases, in fact in about half of the experiments, the injection of red corpuscles saturated with immnne-body mas followed by the development of a certain amount of immune-body, though this was in all cases small as com- pared with the control experiments,-that is, the formation of frwh immune- body was interfered with by the antigen molecules of the corpusclcs being already occupied by immune-body. At the same time, Xeisser and Lubowski (1901 6 )

obtained a similar result on injecting typhoid bacilli saturated with agglutinin, -a certain amount of agglutinin may still be developed in the treated animal.

I n these experiments apparently a certain amount of dissociation must have taken place in order to allow the combined molecules to functionate as antigens by means of their free groups. I n these and similar cases the presence of free antigen is indicated by the anti- substance developed, but in our experiments the separation of immune-body is shown by its effect as a hzmolytic agent. Altmaiin (1 9 12 ’), in a recent paper, has shown that when rabbit’s corpuscles, treated with the corresponding ainboceptor (immnne-serum) from the goat, and repeatedly washed, are injected into the rabbit, various anti- substances to goat’s serum are produced. This might be due to the goat’s immune-body beconling separated froin the corpuscles and acting as antigen, though theoretically i t might act as antigen when still united to the receptors of the red corpuscles (the “antigenic” group being different from that by which it was attached to the corpuscles). We, however, know of no experiments in which the separation of the immnne-body from the corpuscles has been shown by the direct effect of the immune-body,-that is, by its acting again a8 haemolysin in the presence of complement and destroying the corpuscles of the animal used for injection.

METHODS.

In the various experiments the methods followed were practically the same ; details as to the amount of immune-body and corpuscles will be found in connection with the several experiments. The haeniolytic serum for rabbit’s corpuscles was obtained from a goat which had been treated with several injections of rabbit’s corpuscles. The dose of the serum, which had been heated at 55” C. to destroy complement, was 0.01 C.C. for 1 C.C. of 5 per cent. suspension of rabbit’s corpuscles, with normal rabbit’s 6erum as complement ;

94 ROBERT MUIK AND J. W, M'NEE.

i.e. 0.2, C.C. for the corpuscles of 1 C.C. of blood. For each experinlent it

certain quantity of blood, say 10 to 16 c.c., was withdrawn froni the ear vein of a iiormal rabbit and defibrinated ; the hlood was then centrifugalised and the scrum was pipetted off and heated for an hour at 55" C. in order to destroy the complement. The serum was then restored to the corpuscles and tho immune-body added. The mixture was then shaken up and placed in the incubator for an hour at 37" C. ; after tliis time a certain amount of the mixture was injected into the ear vein of a normal rabbit. Sometiines the corpuscles were markedly agglutinated, but this was largely got rid of by shaking ; nevertheless, a fatal result sometimes occurred shortly after the in- jection. I t will thus be noted that the proportion of corpuscles to serum was tho same as in the fresh blood, the difference being that the corpuscles were sensitised with a certain amount of immune-body (never more than a hamolytic dose), and the complement had been destroyed by heating. Test-tube experi- ments showed that (a) the corpuscles separated from the mixture injected mere sensitised, and (b) the serum separated was free from immune-body. Controls were also made by injecting the separated serum into animals with negative results,-no anemia followed. Tho results observed in our experiments must therefore be ascribed to immune-body introduced in combination with red corpuscles.

We shall now give the details of experiments demonstrating the separation of the immune-body injected. In the first two of these the corpuscles were combined with half a haemolytic dose of immune-body -" half sensitised " ; in the. two others with a single haemolytic close -<' fully sensitised." We iiiay add that other experiments were per- formed with similar result.

Experiment 1.

Rannm XIL-Weight, 1595 grms.

Day.

O f . . . . 1 . . . . 2 . . . .

3 , . * .

-

Red Corpuscles.

5,640,000

4,090,000

4,060,000

4,760,000

Lcucocytes.

5,300

8,000

8,000

6,000

Hirnioglobin.

70

59

60

64

Colour Index.

1

1'15

1'18

1

The corpuscles of 10 C.C. of blood combined with 0.9 C.C. of immune-body ( L c . half sensitised) injected intravenously.

In this experiment the fall in the nuniber of red corpuscles was consider- able, namely, 14 millions per c.mm. on the second day, but no haemoglobinuria was noted. On the day after the injection a few nucleated red corpuscles were present in the blood, and also a few basophil megalocytes ; punctate baso- philia was also noted. On the second day after the injection similar changes were found. A second injection of 9 C.C. of half-sensitised corpuscles ww mado on the third day, but death followed at once. The kidneys, liver, and spleen gave a distinct hsmosiderin reaction.

DISSOCIATION OF IMMUNE-BODY. 9 3

12,500 17,000 18,000 15,500 10,600 9,500

6,000 10,300

Day.

90

64 60 60 60 62 60

a4

O f . . . . 1 . . . . 2 . . . . 3 . . . . 4t. . . . 5 . . . . 6 . . . . 7 . . . .

8 . . . .

Experiment 2. RABBIT X1V.-Weight, 1085 grms.

Red Corpuscles.

5,100,000

4,420,000

4,610,000

4,250,000

5,060,000

4,350,000

3,740,000

4,100,000

3,850,000

Leucocytes. H2emoglobin. ~

19,000

14,300

13,800

16,200

... 19,300

23,000

19,000

13,500

60

52

52

54

58

48

42

42

42

Colour Index.

1

1

0.9

1 '1

1

0'9

1

0.85

0 '9

* The corpuscles of 7 C.C. of blood combined with 0.7 C.C. of immune-body

t The corpuscles of 6 C.C. of blood, half sensitised, injected intravenously. (i.8. half sensitised) injected intravenously.

In this experiment a greater degree of anaemia was produced by means of 5 second injection than in Experiment 1. On the day following the first injection distinct hremoglobinuria was present, and a few basophil erythro- cytes, but no megalocytes, were noted. No erythroblasts were present ; there was, however, distinct punctate basophilia. These changes in the blood continued on the two following days. Within an hour after the second injec- tion hmmoglobinsemia was found, and an hour later 10 C.C. of dark crimson urine were passed. On the day following there wa6 distinct haamoglobinuria, but haamoglobinaemia had passed off; on the second day after this injection a few basophil megalocytes appeared in the circulation, and erythroblasts mere also present, some of these approaching the megaloblastic type. Punctate basophilia became more marked in the next few days and many of the cells were over-sized; on the fifth day after the injection some poikilocytosis was present. The animal was killed on this day, and the organs showed a marked iaemosiderin reaction.

Experiment 3. R ~ B B I T xvIr.

Day.

O f . . . . l . . . . 2 . . . . 3 . . . . 4 . . . . 5 . . . . 6 . . . . 7 . . . .

Red Corpuscles.

6,860,000 6,620,000 6,600,000 5,490,000 4,530,000 4,400,000 4,740,000 4,510,000

Leucocytes. 1 Hemoglobin. Colour Index.

1 1 0.86 0 '8 1 1 1 1

The corpuscles of 5 C.C. of blood combined with 1 C.C. of immune-body (i.c. fully sensitised) injected intravenously.

96 ROBERT MUIR AND J W. l%f'NEE.

Within two hours after the injection haenioglobinuria was noted, and thie was still present on the day following ; on the second day nucleated red blood corpuscles were noted-both normoblasts and megaloblasts-and also some basophil megalocytes ; there was, however, no punctate basophilia. These changes continued for some days. The animal was killed on the twelfth day, and the usual iron reaction was noted in the organs.

Experiment 4.

RABBIT XXYVI1.-Weight, 2170 grms.

Day.

0 ' . . . . I t . . . . 2 . . . s . . . . 4 . . . . 6 . . . .

Red Corpuscles. Leucocytcs.

6,900,000 6,000

6,200,000 8,400

5,860,000 12,000

5,010,000 9,000

4,600,000 5,600

4,960,000 18,000 .

. -~ - ._ - ..

Hoemoglobin.

48

42

- . . ~ . - .

Colour Index.

1

1

0 '9

0 *9

0 '8

0 '8

. . The corpuscles of 6'6 C.C. of blood combined with 1'8 C.C. of inimune-body

t The corpusclen of 6 C.C. of blood, fully sensitised, injected intravenously. (i.e. fully aensitiied) injected iutravenoualy.

111 this e x p e r u e n t ~1 rcarlced degree of anaema wcw produced by two injections on succeeding days. On the day after the first injection there was dist.inct haemoglobinnria, and on tlic day after the second injection this was again well marked, and hEmog1obitmmia was also present (this had not been tested for before). Films of blood of the day after the second injection showed the presence of some sliadoms in the circulation, n few basophil erythrocytes of ordinary size, and a few normoblasts ; a few small leucocytes with pyknotic nuclei were also noticed. On the next day megalocytes appeared, and on the following days became more numerous. The animal was killed on the fourth day after the second injection, and the orgniis gavc an extreme i r h reaction.

I n order to determine whether the anaemia produced by the injection of the sensitised corpuscles was real or not, the blood was washed out and the total volume estimated by the direct method. Tho volume of blood was thus ascertained to be one twenty-sixth of the body weight, that is, i t was not increased; accordingly, the fall in t h e number of corpuscles per c.nim. indi- cated a true anaemia.

We may now summarise the results of these experiments and consider their significance. The sensitised corpuscles injected undergo lysis within a short time, with resulting hzmoglobinzmia and hznio- globinuria, the former being detectable within one hour after the injection ; following upon this rapid solution of corpuscles, which is what one might expect, a distinct degree of anamia develops, and this is characterised not only by the fall in the number of corpuscles per

DISSOCIATION OF IMMUNE-BODK 9 i

c.mm., but also by the morphological changes in the circulating blood. It has also been shown, by washing out the blood and estimating the volume directly, that there is no increase in the volume, and that accordingly a true anaemia is present.

The fall in the number of the red corpuscles is remarkable, and is in fact greater than what would correspond to the amount of lysis in vdtro if the immune-body had been used in the free condition. Thus in Experiment 1 the amount of blood destroyed in addition to the lysis of the injected corpmcles was about 16 c.c., whereas the amount of immune-body injected (0.9 c.c.) would have lysed only 5 C.C. of blood im vitro ; and in Experiment IV., the amount destroyed as the result of two injections was fully 30 c.c., whereas the corresponding amount in mtro would have been only 12.5 C.C. if the immune-body had been free. The maximum fall in the number of red corpuscles is not reached till a day or two after the injection,-in two experiments it occurred on the second day, in one on the third day, and in another on the fourth day. With regard to these two points-namely, the amount of blood destruction and the time necessary for its occurrence -the present experiments correspond with those already published, in which the immune-body was injected in the free condition ; although in the present series the actual amount of anaemia is of course much less. As we have stated, in addition to the actual fall in the number of red corpuscles, morphological changes occurred in all the Bxperi- ments ; these included polychromatophilia, especially in megalocytes, punctate basophilia which was very marked in two experiments, the presence of erythroblasts in the circulating blood, variations in the size of the corpuscles, and in one case slight poikilocytosis. All these changes correspond with those already described by us as pro- duced by a hremolytic serum, though they are less in degree. The only important difference is the condition of the colour index, ,which was scarcely ever raised above normal. This may be due to the smaller degree of the anaemia or the slower action of the poison. In all experiments a marked harnosiderin reaction was given by the liver, spleen, and kidneys, but this was no doubt in part due to the hzemoglobin derived from the corpuscles injected.

The various changes described above indicate clearly the action of a haemolytic poison, and this we consider must have been the immune-body injected in union with the corpuscles. It .has been suggested that substances derived by the breaking down of red corpuscles might be concerned in the result, but there are no facts in favour of this, and we have found that the injection of a similar amount of corpuscles to that used in the experiments, laked previously with a trace of saponin, had no effect in lowering the blood count on the days following the injection. After the sensitised corpuscles have undergone solution the immune-body probably at first remains attached to the stromata,-at least, if we can judge from test-tube

7 4 01 PA?&-VOL XVII.

98 DISSOCIATION OF IMMUNE-BODX

experiments. One of us (1903 s, has shown that when red corpuscles containing multiple doses of immune-body are lysed in witl.0, the immune- body remains in union with the receptors in the stromata. This is shown by the fact that it is not taken up by fresh corpuscles a t 0" C. as it would be if it were free, though it is dissochble a t higher temperatures. But in the case of red corpuscles containing a single dose (as was used in our experiments) no dissociation can be demonstrated G Uitro, probably because the amount separated is too small. Within the living body, however, the results obtained force us to the conclusion that the immune-body introduced, not only lyses the corpuscles with which it was combined, but afterwards leads to the destruction of a considerable amount of blood, and we are of opinion that the immune- body is split off by some agency to a greater degree than occurs by simple physical dissociation.

REFERENCES.

1. MUIR AND M'NEE . . . Journ. Path. and Bacterial., Cambridge, 1912,

2. MORQFXROTH . . . . . Munchen. med. Wc7mchr.,.?903, No. 2. 3. MUIR . . . . . . Lancet, London, 1903, vol. 11. p. 146 ; ' I Studies

4. TON DUNQERN . . . . Munchen. med. Vchnschr., 1900, No. 20. 5. SACHS . - . . . . . Centrallrl. f. Bakteriol. u. Parasitenk., Jena,

6 . NEISSER UND LIJBOWSKI 7. ALTXANN . . . . , . Ztschr. f. Inznzunitut~forschun~, 1912, Bd.

vol. xvi. p. 410.

on Immunity," London, 1909.

1901, Bd.xxx. S. 491. . Bid . , 1901, Ed. xxx. S. 483.

xiii. S. 219.