THE RELATIONSHIP BETWEEN THE H B M O - LYTIC COMPLEMENT AND THE OPSONIC POWER OF NORMAL GUINEA-PIG SERUM.

JOHN GORDON, HUGH ROBINSON WHITEHEAD and ARTHUR WORMALL. From the Departments of Pathology and Bacteriology and of Physiology, the

School of Hedicine, Leeds, and the Department of Bacteriology, College of Medicine, Newcastle-on-Tyne.

TEE relationship between the opsonic power of normal serum and the power to hsemolyse sensitised red corpuscles has been studied by numerous authors and in many instances evidence has been produced to show that the opsonic action of a normal serum involves the action of complement or perhaps a more complex system consisting of a thermostable substance and a thermolabile component of the com- plement type. If the latter view is correct, the opsonic system may be regarded as being similar to the hzemolytic system in consisting of a relatively heat-stable and probably specific amboceptor and a heat- labile non-specific complement.

M.uir and Martin (1906) produced evidence to show that there is a close similarity between normal thormolabile opsonins and haemolytic complement, for they found that substances which remove hzemolytic complement from normal guinea-pig serum also remove the opsonic power. The absorbing substances used for this purpose were (a) sensitised red cells ( b ) serum precipitates formed by mixing antigen and antibody and (c) sensitised bacteria, and in all cases the absorption of hsmolytic complement and opsonins were closely parallel. Bkcher (1907) and Levaditi and Inmann (1907) produced further evidence pointing to a similarity between normal opsonin and complement, whilst Neufeld and Hiine (1907) showed that treatment of normal serum with yeast cells removes both hemolytic complement and opsonin.

Other authors however have expressed the opinion that a thermostable opsonic constituent (amboceptor) is concerned, together with complement, in opsonic action. Dean (1907) showed that the opsonic power of heated immune serum was greatly increased by the addition of a small amount Qf unheated normal serum. Cowie and Chapin (1907, 1) obtained similar results with normal human Serum and found that the addition of a small amount of normal human serum could restore the opsonic power of human serum which had been heated at 55" C. for ten minutes. From these experiments the conclusion was reached that the opsonin of normal human serum has a double amboceptor-complement structure like hamolysins and bacteriolysins. Further evidence in favour of this view was presented in a later paper (Cowie and Chapin 1907, 2), when it was shown that Staphylococcus treated with heated serum and then washed is more readily opsonised by a normal serum than is the untreated organism. I n addition, treatment of normal human serum with staphylococci results, under certain conditions, in the loss of practically all the opsonic power but leaves behind

57

58 3. GORDON, H , R. WHITEHEAD AND A. WORMALL

a substance which can reactivate a heated serum for opsonic activity. The same authors (1907,3) submitted further evidence in support of the amboceptor- complement view of opsonic action. They found that Staphylococcus, treated with normal human serum at 0°C. and then washed with cold salt solution, is not much more susceptible to phagocytosis in the absence of senun than the untreated organism, but is more readily opsonised by a dilute normal serum or by a serum which had been inactivated by contact with staphylococci in the cold. Thus at 0" C. the bacteria absorb the amboceptor component only of the opsonic system, Browning (1925) states that the taking up of opsonins at low temperatures without the removal of complement may be attributed to the existence of a natural immune-body which plays a part in the opsonic effect, leading to the union of complement, or it may be due to the existence of multiple complements.

It is possible to study what rHe, if any, is played by complement in opsonic action by observing the effect on the opsonic power when complement is " split " into fractions, or when one or more components are inactivated. Simon, Lainar and Bispham (1906) found that the opsonin of serum is associated with the globulin fraction and Heinemaiin and Gatewood (1912) similarly found that the opsonins of antistreptococcic and antigonococcic sera are precipitated with the globulins. Ledingham and Dean (1912), however, showed that mid-piece as prepared by the CO, method was unable by itself to sensitise B. typhosus for phagocytosis.

I n an investigation of the properties of hsemolytic complement the present authors have shown (1926, 1) that small amounts of ammonia inactivate haemolytic complement by the almost specific destruction of a component which is relatively heat-stable and which is associated with the albumin fraction of the serum. Thus ammonia-treated serum has no hamolytic action on sensitised red cells but is reactivated by the addition of serum heated a t 56" C. or heated "end-piece" (albumin fraction). It has been shown later (Gordon and Wormall 1928) that ammonia has a similar action on the bactericidal power of normal guinea-pig serum against B. dyysenterice (Flexner), and it was concluded that this bactericidal system consists of haemolytic complement and an immune-body present in the normal serum. Preliminary investigations of a similar nature (Gordon, Whitehead and Wormall, 1926, 2) showed that the fourth component of haemolytic coniplement is not essential for opsonic action, and the investigations described below form an extension of this observation.

Treatment of normal guinea-pig serum with small amounts of ammonia results in the complete loss of haemolytic action towards sensitised red cells, due to the inactivation of the fourth component of complement, but no corresponding loss of opsonic power occurs. Thus the conclusion is reached that although the other components of hsemolytic coniplement may function in the opsonic system, the fourth component is not essential.

The treatment of serum with bacteria usually results in the removal of both haemolytic coniplement and the opsonic power, and this is quoted in support of the view that the opsonic system of normal serum is a coniplement or arnboceptor-complement system. If suitable

COMPLEMENT AND OPSONlN 59

amounts of the organism (Staphylococcus auTeus in these experiments) are added to normal guinea-pig serum and the organisms removed by centrifuging after incubation of the emulsion for about 2 hours, it is found that the serum has no opsonic action and no hamolytic action on sensitised red cells. The centrifuged bacteria however, after being washed with 0.9 per cent. NaCl solution, are readily phagocyted, and the loss of opsonic power by the serum is attributed to the absorption or adsorption of the opsonins by the bacteria. If the view expressed above is correct-that the fourth Component of complement is not essential for opsonic action-then the fourth component of com- plement need not necessarily be absorbed by the bacteria, and indeed the non-removal of this component by the bacteria would furnish evidence in support of the hypothesis. Experiments carried out to test this point have shown that treatment of the serum with bacteria under conditions which lead to the complete loss of hmnolytic and opsonic functions does not cause the removal of the fourth component of complement from the serum. The bacteria, which are removed from the serum by centrifuging, are however fully sensitised for subsequent phagocytosis, and the conclusion is drawn that these bacteria have removed the whole of the opsonic system; this system therefore does not include the fourth component of complement.

Experimental. To determine the opsonic power of the normal or treated guinea-pig serum

0.1 C.C. of an emulsion of Staph?ylococcus aureus was added to 0'5 C.C. of the serum in a centrifuge tube; the mixture was then incubated a t 37°C. for 1 hour and washed guinea-pig leucocytes added. After a further incubation for 1 hour, films were made, stained and examined for phagocytosis. For our purpose it sufficed to determine only the presence of a good phagocytosis or the absence of phagocytosis and it was not found necessary to make opsonic counts.

The hemolytic complenient activities were determined by diluting the sera with 0.0 per cent. NaCl solution and adding various amounts of the diluted sera to 1 C.C. of 4 per cent. sensitised ox cells. The total volume in each tube was adjusted to 2'0 C.C. where necessary by the addition of 0'9 per cent. NaCl solution, and the tubes were then incubated a t 37°C. The amount of haemolysis after incubation for hour, & hour and 1 hour was recorded thus :

+ + + + complete hzemolysis. - no hzemolysis. + + +, + + or + intermediate grades of haemolysis.

TILO action of ammonia on the opsonin and complement of nornzal guinea-pig se~um.

I n the earlier experiments normal guinea-pig Beruni was treated with one-quarter of its volume of N16.5 NH, solution, but it was found later that treatment with a smaller volume of stronger ammonia solution was preferable for the opsonic experiments. To inactivate the hmnolytic complement, 3 C.C. of guinea-pig serum were treated with 0.05-0.10 C.C. of 0.87 N . NH, solution and the mixture incubated a t 37°C. for 14 hours. The serum was then neutralised by the addition

60 3. GORDON, H. R. WHITEHEAD AND A. WORMALL

of the necessary amount of N . HC1 (determined by the titration of an aliquot portion) and then tested for opsonic and hwmolytic action. The following is a typical result from a large number of experiments in which the amount of NH, used and the time of incubation were varied.

Opsonic power.-No differences could be detected between the opsonic powers of normal and ammonia-treated sera. Controls with 0'9 per cent. NaCl solution showed no phagocytosis.

Bamolytic activity- Hsmolysis of sensitised red cells after incubation for 1 hour :- 1.0 C.C. normal guinea-pig serum (1 : lo) . ++++ 0 5 C.C. ,, Y, ,, (1 : 10) . ++++ 1.0 C.C. NH,-treated guinea-pig serum (1 : lo) . - 0'5 C.C. ,, 3 , ,, (1 : lo) . - 0'5 C.C. ,, serum (1 : 10) plus 0'5 C.C. heated serum . + + + +

The heated serum was guinea-pig serum heated a t 56" C. for 30 minutes and

These experiments show that destruction of the fourth component of complement by ammonia results in a complete loss of hemolytic action for sensitised cells, whereas no detectable loss of opsonic power occurs.

Removal of hmnoly t ic cowplement and the opsonins of normal guinea-pig serum by bacte&.

A heavy emulsion of Staphylococcus awreus was added to nornial guinea-pig serum and the mixture incubated a t 37" C. for 2 hours with frequent shaking. The bacteria were then removed by centrifuging for 1 hour a t high speed, and the opsonic and haniolytio powers of the supernatant serum determined. The NH,-treated serum was prepared as described for the preceding experiment.

then diluted with 9 volumes of 0'9 NaCl solution.

Normal guinea-pig serum . . Good. Opsonic activity NII,-treated guinea-pig serum . Good. . None. The bacteria removed from the last serum were washed with 0'9 per cent.

IIcemolytic activity.-All the sera were diluted with 9 volumes of 0.9 per cent.

1 Serum treated with bacteria .

NaCl solution and were found to be readily phagocyted in NaCl solution.

NaCl solution. Hsmolysis of sensitised red cells after incubation for 1 hour :- 1.0 C.C. normal guinea-pig serum . ++++ 0'5 C.C. ,, 3 , ,, . + + + + 1.0 C.C. NH,-treated guinea-pig serum . - 0'5 C.C. 1, , l *

0 5 C.C. ,, serum + 0'5 C.C. heated serum . . ++++ 0'5 C.C. 9 1 ,, + 0'5 C.C. bacteria-treated serum . + + + + 0 5 C.C. 9 9 ,, + 0'6 C.C. heated bacteria - treated

1-0 C.C. bacteria-treated serum . -

3, -

serum . . +++ or + + + + Y, - 0'5 C.C. 79

COMPLEMENT AND OPSONZN 61

From these results it will be seen that neither NH,-treated serum nor serum treated with bacteria had any hsmolytic complement activity. When combined, however, they show strong hamolytic action towards sensitised red cells. Thus the treatment of normal guinea-pig serum with Staphylococcus aureus in amounts sufficient to remove the opsonic power does not result in the removal of the fourth component of complement.

Summary.

Destruction of the hzemolytic complement activity of normal guinea-pig serum by small amounts of ammonia does not reduce to any significant extent the opsonic power of the serum for Staphylococcus aurew. Incubation of the iiormal serum at 3'7°C. for 2 hours with this organism in amounts sufficient to remove the opsonic and hzemolytic complement activities does not remove the fourth component of complement. The conclusion is drawn from this evidence that one component of complement a t least, the fourth component, is not essential for the opsonic action of the normal serum.

Two of the authors (J. a. and A. W.) desire to thank the Medical Research Council for part-time personal grants and for grants which have partly defrayed the expenses of this research.

REFERENCES. BACHER , . . . , . . , . 2. fur Hyg., 1907, Ivi. 33. BROWNING, . . . . . . . Immunochemical studies, London, 1925. COWIE AND CHAPIN (1) . . . J . Med. Res., 1907, xvii. 57.

$9 ,, (2) . . . Ibid., 1907, xvii. 95. 9, ,, (3) . . . Ibid., 1907, xvii. 213.

DEAN . . . . . . . . . Brit. illed. Journ., 1907, ii. 1409; Proc. Roy.

GORDON, WHITEEEAD AND Biochem. Journ., 1926, xx. 1028.

GORDON, WHITEHEAD AND Ibid., 1926, xx. 1044.

Soe. London, 1907, lxxix. B, 399.

WORMALL (1)

WORMALL (2) GORDON AND WORMALL. . . this JOUT'nal, 1928, XXG. 753. HEINEMA" AND GATEWOOD . J. Infect. Dis., 1912, x. 416. LEDINGBAM AND DEAN . . . J. H?ygzelze, 1912, xii. 152. LEVADITI AND INMANN . . . Comp. rend. SOC. Biol., 1907, Ixii. 683, 725, 817

and 869. MUIR AND MARTIN . . . . Brit. Med. Jown., 1906, ii. 1783. NEUFELD AND HUNE. . . . Arb. a. d. Kais. Gesundh., 1907, xxv. 164. SIMON, LAMAR AND BISPHAM J. Exper. Med., 1906, viii. 651.