Immunology 1979 37 881

Complement activation by aluminium and zirconium compounds

V. D. RAMANATHAN, P. BADENOCH-JONES & J. L. TURK Department of Pathology, Royal College of Surgeons of England, Lincoln's Inn Fields, London

Acceptedfor publication I March 1979

Summary. The activation of complement by a number of metal compounds, previously found to induce chronic inflammation, was investigated. Results obtained were compared with complement activation by inulin and zymosan. It was shown that complement activation by these metal compounds did not necessarily involve either the classical or the alternative pathways. Some of these compounds were unable to activate complement in the absence of detectable plasminogen. The results obtained indicate a relationship between the ability of these compounds to induce chronic inflammation in the guniea-pig and to activate complement.

Parker, 1978). Granuloma formation in tissues following the introduction of colloidal alum-precipitated proteins is the basis for the use of some of these compounds as adjuvants in the immune response (Proom, 1943). In addition, it is known that certain aluminium compounds can be extremely toxic on inhalation and induce pneumoconiosis (Lynch & McIver, 1954; Perry, 1963). Sodium zirconium lactate is capable of producing an allergic type of granuloma in the skin of man (Shelley & Hurley, 1958) and guinea-pigs (Turk & Parker, 1977b). This type of lesion has been shown to contain giant cells and epithelioid cells (Black & Epstein, 1974; Turk, BadenochJones & Parker, 1978) similar to those observed in sarcoidosis. Schorlemmer, Bitter-Suermann & Allison (1977) have shown a parallel between the induction of chronic inflammation by some compounds with their ability to release lysosomal enzymes from macrophages and the activation of the alternative pathway of complement. This communication describes the results of an investigation of the ability of the aluminium and zirconium compounds that cause granuloma formation and macrophage damage to activate the complement pathway.

INTRODUCTION In previous reports from this laboratory, a number of aluminium and zirconium compounds have been shown to induce chronic inflammation (Turk & Parker, 1977a) and to release lactate dehydrogenase from cultured macrophages (Badenoch-Jones, Turk & Abbreviations: ACH: aluminium chlorohydrate gel;

*AI(OH)3: aluminium hydroxide gel; AT: anaphylatoxin; EDTA: ethylenediaminetetraacetic acid, disodium salt; NaZrL: sodium zirconium lactate; NHS: normal human serum; THC: total haemolytic complement; VBS: veronalbuffered saline; ZAG: zirconium aluminium glycinate gel; Zr(OH)4: zirconium hydroxide gel. Correspondence: Professor J. L. Turk, Department of Pathology, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN. 00 19-2805/79/0800-088 1$02.00 () 1979 Blackwell Scientific Publications

MATERIALS AND METHODS Metal compounds Kaolin and aluminium hydroxide were purchased 881

882

V. D. Ramanathan, P. Badenoch-Jones & J. L. Turk

from Hopkin & Williams, Romford, Essex. Basic zirconium carbonate was supplied by the British Aluminium Co. Ltd, Chalfont Park, Bucks., and NaZrL,

to determine the levels of plasminogen were obtained from Behringwerke, Marburg, Germany.

ACH and ZAG were supplied by the Procter & Gamble Company, Cincinnati, U.S.A. NaZrL is soluble in normal saline (final pH 6-8). Kaolin was washed three times in normal saline and was resuspended at a final concentration of 100 mg/ml. The other metal compounds were converted to the same physical form as described previously (Badenoch-Jones et al., 1978) and resuspended at a final concentration of 20 mg/ml.

Buffers Veronal-buffered saline pH 7-2, veronal buffer containing 0-01 M EDTA pH 8-6 and 0 05 M phosphate buffer pH 7-5 were used.

Complement Pooled normal serum from laboratory volunteers was used as the source of complement. Serum was aliquoted in 1 ml quantities and stored at - 700, and was used only once after thawing. Partially purified anaphylatoxin was prepared as described by Kleine, Poppe & Vogt (1970).

Plasminogen-depleted serum This was obtained by passing fresh NHS through a column of lysine-Sepharose 4B as described by Deutsch & Mertz (1970). As a control, an equal volume of serum was passed through a Sepharose 4B column. Levels of plasminogen, total serum proteins and THC were measured in samples of sera that did not pass through any column and those that were passed through Sepharose and lysine-Sepharose columns. Guinea-pigs Male guinea-pigs (350-400 g) were purchased from A. Tuck & Son Ltd, Rayleigh, Essex, and starved for 24 h prior to use.

Reagents The following reagents were obtained as indicated. EDTA from British Drug Houses, Poole, Dorset; Folin's reagent, histamine, inulin and zymosan from Sigma, Poole, Dorset. Zymosan was prepared as described by Fine, Marney, Colley, Sergent & Des Prez (1972). Lysine-Sepharose and Sepharose 4B was obtained from Pharmacia, Uppsala, Sweden; agarose from L'Industrie Biologique Francaise, Paris; sheep erythrocytes from Gibco Bio-Cult, Paisley, Scotland; anti-sheep haemolysin from Difco Laboratories, West Molesey, Surrey, and anti-Plc/ptA antiserum from Miles Laboratories, Slough, Bucks. M. partigen plates

Total haemolytic complement Samples of serum were treated with VBS pH 7-2 or metal compounds for 15 min at 37°. After centrifuging off the particulate compounds, serum was diluted to 1 in 100 and the residual THC was measured as described by Mayer (1961).

Anaphylatoxin assay The generation of AT was measured by recording contractions of the isolated terminal ileum of guineapig. A 3 cm strip of ileum was suspended in a 10 ml bath of Tyrode's solution maintained at 370 and gassed with oxygen. The isotonic movement of the guinea-pig ileum was recorded with a pen recorder. To eliminate the effect of tachyphylaxis, four-point assays were performed (Rocha, Silva & Rothschild, 1956).

Two-dimensional electrophoresis Monospecific anti-C3 antiserum was used in the twodimensional electrophoresis system to demonstrate the splitting of C3 and the formation of C3b (Hudson & Hay, 1976). Percentage conversion of C3 to C3b was measured from projections ofthe original electrophoresis plate using planimetry. Total protein estimation Total serum proteins were measured with Folin's reagent using bovine serum albumin as the standard (Lowry, Rosebrough, Farr & Randall, 1951).

RESULTS Consumption of THC by kaolin, ACH, ZAG, Zr(OH)4 and NaZrL The metal compounds were tested for their ability to consume THC at several doses. In all experiments, a

metal compound was incubated with an equal volume of NHS at 370 for 15 min, followed by centrifugation and measurement of residual THC in the supernatant. The results are expressed as percentage loss of complement activity compared with NHS treated with VBS.

I883

Complement activation by Al and Zr compounds 100 _

80 _

60 0

40

o

10

0

20

30

40

50

mg /mL

(0 100

(

00 0 625

bI)t

1*25

2-5

5-0

mg/mL

80

Figure 2. Removal of total haemolytic complement from normal human serum by sodium zirconium lactate, a soluble metal compound.

60' 40

20

0

Figures 1 and 2 show that these compounds consume complement in a dose-dependent manner. Kaolin, ACH and ZAG were unable to consume complement when incubated with NHS at 40, whereas Zr(OH)4 and NaZrL were able to do so (Table 1). To see whether this consumption of complement was through the classical or the alternative pathway, NHS was treated with the metal compounds in the presence of EDTA. The results suggest that the possible mechanism of action was neither through the classical nor the alternative pathway (Table 2). It was found that all these compounds were able to

-

...

2X5

5X0

7'5

10.0

mg/mL

Figure 1. Removal of total haemolytic complement (-) and protein (-- -) from (a) normal human serum by kaolin and (b) metal compounds, v ACH, * ZAG, * Zr(OH)4. All of these compounds are insoluble. Each point represents the mean of four replicates.

Table 1. The effects of dilution and temperature on the loss of THC induced by various substances

Concentration mg/ml of Challenge undiluted serum Zymosan Kaolin ACH ZAGS ZrOH Inulin NaZrL

4 80 15 20 20 40 10

Loss of complement (%) at serum dilution of

Loss of complement (%)

1:2

1:100

at 40

at 370

100 84 100 100 100 50 100

0 71 100 100 100 0 100

0 0 10 5 81

100 78 100 100 100

100

100

V. D. Ramanathan, P. Badenoch-Jones & J. L. Turk

884

Table 2. The effect of EDTA on the loss of THC induced by various substances Loss of complement (0)

Chelator None EDTA lOmm

Challenge EA* 2 x 109 cells/ml Zymosan 2 mg/ml Kaolin 40 mg/ml ACH 10 mg/ml ZAG 10 mg/ml ZrOH lOmg/ml Inulin 20 mg/ml NaZrL 10 mg/ml

100 100 82 100 100 100 50 100

Serum

ACH

Seru

AC

*Sheep erythrocytes coated with optimum amount of haemolysin

consume complement when treated at a serum dilution of 1 in 100, at which dilution the alternative pathway factors are inactive (Table 1).

Consumption of THC by AI(OH)3 It was found that AI(OH)3 did not consume complement avidly in undiluted serum, removing a maximum of 30%, whereas addition of 10 mm EDTA potentiated this action (Fig. 3). At a serum dilution of 1 in 100,

c

E

aJ

f

-J CL

E

0 0

J

5.0

20.0 10.0 Al (OH)3 mg / ml of undiluted serum

Histamine I Opg

Histamine 1-0 pg

0 0 68 100 100 100 0 100

300

Figure 3. The effects of serum dilution and EDTA on the removal of total haemolytic complement by aluminium hydroxide. Serum complement is not removed by aluminium hydroxide in the 1:2 diluted serum (-), to such an extent as in 1:100 diluted serum (o). Addition of 10 mM EDTA to 1:2 diluted serum potentiates this removal of complement( x ). The removal of protein, by aluminium hydroxide, from 1:100 diluted serum is also shown (---).

Serum

J

ACH

IE -Time Figure. 4. Tachyphylaxis: the decreasing response of the ileal strip to repeated challenges with serum treated with ACH.

however, complement consumption was comparable to that of the other metal compounds. Protein building ability The amount oftotal protein present in the supematant after treatment of NHS with the metal compounds was estimated and the results expressed as percentage removal of proteins compared with serum treated with VBS (Figs 1 and 3). This was done to exclude a nonspecific removal of proteins and to find out whether there was any correlation between the ability to bind proteins and to activate complement. It was found that all the particulate metal compounds bind proteins, though there was a definite dissociation between the removal of complement and proteins. This suggested that non-specific removal of proteins could not account for the amount of complement consumed. The possibility of a specific removal of complement components could not, however, be excluded.

Anaphylatoxin assay AT assay using strips of the terminal portion of guinea-pig ileum was performed to demonstrate the

Complement activation by Al and Zr compounds

885

Serum + zymosan

Serum + buffer

....

_..Ak

Figure 5. Two-dimensional electrophoresis demonstrating the formation of C3b in serum treated with zymosan, ACH and ZAG.

formation of C3a and C5a. Partially purified AT was prepared from serum treated with zymosan and metal compounds. One hundred microlitres of the preparation induced contraction of the ileum. This contraction was thought to be due to AT because (a) it proTable 3. Anaphylatoxin production by various compounds in the presence or absence of 10 mm EDTA

Contraction of guinea-pig ileum as a percentage of contraction induced by 0-1 pg histamine*

Compound added to serum

Zymosan Inulin Kaolin ACH

A1(OH)3 NaZrL ZAGS Zr(OH)4

Chelator EDTA 10 mM

mg/ml

Nil

2 20 50 10 15 5 10 10

78-8 + 5-3 88.3 + 10-4 78 3+21 3 106-1+172 90 7 + 21 6 80-7 + 14 4 93-5+11-9 0

0 0 88 3+10 7 753+10-8 106 7 +15-0 77 3 + 11 0 92-8+13.1 0

*Mean + standard error of four replicates.

produced tachyphylaxis (Fig. 4), (b) the contraction was inhibited by antihistamine and (c) it was not produced following inactivation of serum at 560 for 30 mins. Further, in the case of zymosan and inulin, which were used as controls, this factor was not produced after the addition of 10 mm EDTA to the serum. Using four-point assays, the contractions produced were measured and compared with those induced by 0.1 ig histamine and the results are expressed as a percentage of the latter (Table 3). It was found that the metal compounds, with the exception of Zr(OH)4, produced AT and that this production was not blocked by the addition of 10 mm EDTA to the serum prior to challenge.

Two-dimensional electrophoresis Two-dimensional electrophoresis using anti-C3 antiserum was performed to show the formation of C3b (Fig. 5). This confirmed the results of the earlier experiments showing that all the metal compounds except Zr(OH)4 activate complement and that this is temperature-dependent but not blocked by 10 mM EDTA (Table 4).

886

V. D. Ramanathan, P. Badenoch-Jones & J. L. Turk Total serum protein

Total haemolytic complement

Plasminogen

100 0

E

80 -

0

60 F-

0

40 p

0

IH

C

I.

20 -

Figure 6. Removal of total serum protein, total haemolytic complement and plasminogen from serum passed through a Sepharose-lysine column (stippled) and a Sepharose column (hatched). Results are expressed as a percentage of normal serum values (open).

Experiments to detennine the pathway of activation As the results of the blocking experiments with EDTA demonstrate, the formation of the classical or the alternative pathway convertases is not necessary for the activation of complement by the metal compounds. Kaolin is known to activate plasminogen (Ogston, Ogston, Ratnoff & Forbes, 1969), which when converted to plasmin becomes a powerful proteolytic enzyme. Using lysine-Sepharose column, it is possible to remove plasminogen selectively from serum (Deutsch & Mertz, 1970). The plasminogen content of the

serum was

checked before and after

so. The reduction in the plasminogen content of the serum passed through the Sepharose column is due to dilution, as the total protein and complement levels also show a corresponding decrease. Serum that had been passed through a Sepharose or a lysine-Sepharose column, was treated with the metal compounds and two-dimensional electrophoresis was

performed (Table 4). It is clear that ACH, Al(OH)3 and ZAG are dependent on plasminogen to activate C3, whereas kaolin and NaZrL are not. Zymosan is able to activate C3 in the plasminogen-depleted serum, showing that the complement components are intact.

through the column using immunodiffusion plates containing antiserum against plasminogen. As a control, an equal volume of NHS was passed through a Sepharose 4B column. In addition to plasminogen levels, total serum protein and THC were also measured. As Fig. 6 demonstrates, lysine-Sepharose removes plasminogen, whereas Sepharose does not do passage

DISCUSSION

The results described above demonstrate that all the metal compounds studied, except Zr(OH)4, activate complement resulting in the production of anaphyla-

Table 4. Formation of C3b by various metal compounds

NHS passed through NHS passed through AT40 NHS+Buffer NHS+EDTA 10mm Sepharose4Bcolumn lysine-Sepharosecolumn

Buffer Zymosan ACH Al(OH)3 ZAG NaZrL Kaolin Zr(OH)4

0 0 0 0 0 0 0 0

0 59 8 ±7-6 28-4+ 2-6 36-7+ 2-6 38-9+ 4 5 55 2+ 1-7 27-5+12-7 0

0 0 33-5+ 8 5 36-5 + 3-2 36-2+ 5 8 36-2+ 0-8 40-9+11-1 0

0 58 4+8-1 31-1+4-1 39-2+ 3-7 43-2+9-2 44-8+ 5-3 29-4+7-1 0

0 57-7 +±30 0 4-1 +2-6 0 28-8 + 8-3 349+6 5 0

Formation of C3b was demonstrated by two-dimensional immunoelectrophoresis as described in the text. Results are expressed as percentage of conversion of C3 to C3b. Each value represents mean + SEM of four replicates

Complement activation by Al and Zr compounds toxins and C3b, and that this activation is neither through the classical nor the alternative pathway. Kabat & Mayer (1961) found that kaolin produces AT and Polley & Nachmann (1975) have shown that AI(OH)3 gel consumes complement. These latter authors reported this as an incidental finding in an experiment where AI(OH)3 gel was used to adsorb prothrombin from plasma. They found that AI(OH)3 consumed up to 50% of complement when incubated with plasma at 370 but not at room temperature, whereas the adsorption of prothrombin occurred at both temperatures. The mechanism by which AI(OH)3 may be anticomplementary, however, was not discussed by these authors. Lachmann (1975) suggested that kaolin, like latex, could aggregate immunoglobulins on its surface and thereby activates complement, but the results of the blocking experiments with EDTA reported here suggest the involvement of additional mechanisms. As kaolin is known to activate plasminogen (through the Hageman factor; Ogston et al., 1969), which in turn can activate CIs and C3 (Ratnoff & Naff, 1967; Ward, 1967) we thought that this might be the pathway of activation. Results of the experiments using plasminogen-depleted serum demonstrate that while ACH, Al(OH)3 and ZAG are dependent on plasminogen for their activation of C3, kaolin and NaZrL are not. Experiments are now in progress to find out whether ACH, Al(OH)3 and ZAG activate plasminogen directly. It is not known why AI(OH)3 does not consume THC in undiluted serum as efficiently as in diluted serum, though it activates a considerable amount of C3 (36%) in undiluted serum. Kaolin and NaZrL activate C3 even in the absence of plasminogen and we do not have an explanation for this phenomenon. It is possible that C3 is directly activated by kaolin and NaZrL. Alternatively, as has been suggested in the case of radiographic contrast media (Till, Rother & Gemsa, 1978; Kolb, Lang & Lasser, 1978), kaolin and NaZrL might bind and remove protease inhibitors and thereby activate complement. All the metal compounds described above, with the exception of Zr(OH)4 have been shown to induce chronic inflammation. The induction of pneumoconiosis (Lynch & McIver, 1954) and foreign body granulomas (Lord & Wilson, 1968; Reed, Valerio, Ulland, Valerio & Stookey, 1970) by kaolin has been well documented. in addition, kaolin is known to be haemolytic (DePasse & Leonis, 1974). In a previous report (Badenoch-Jones et al., 1978) it was shown that

887

ACH, AI(OH)3 and ZAG were haemolytic, toxic to macrophages and induced foreign body granulomas, whereas Zr(OH)4 was inactive. It is of interest to note that Zr(OH)4 which is nontoxic to macrophages in vitro and which does not induce granuloma formation in the skin of the guineapig, does not activate complement. It consumes THC even at 40, although the protein removal pattern does not suggest a non-specific removal of complement. As with concanavalin A (Bitter-Suermann, Hadding, Schorlemmer, Limbert, Dierich & Dukor, 1975), however, the possibility of binding of one or more complement components without activation cannot be excluded. In the case of asbestos, a positive correlation has been shown between the ability of different types of asbestos to adsorb proteins and to cause chronic inflammation (Morgan, Holmes & Talbot, 1977). Similarly, in the case of radiographic contrast media, the strength of protein binding has been found to correlate with the toxicity as measured by the LD_o (Lasser, Farr, Fujimagari & Tripp, 1962). In our study there was no correlation between the ability of the various compounds to remove proteins and to activate complement, though kaolin which removes less than 1 1% of proteins is the least anticomplementary compound on a weight basis. With the exception of NaZrL, all other compounds occur in the form of complexes and therefore cannot be compared on a molar basis. Thus complement was activated by all the metal compounds that induced chronic inflammation. This is in agreement with the general findings of Schrolemmer et al. (1977). As we have shown, however, this activation need not be through either the 'classical' or the 'alternative' pathways, but for some of the compounds may be through a pathway involving plasminogen. ACKNOWLEDGMENTS This study was supported, in part, by the Procter & Gamble Company, Cincinnati, U.S.A., and the British Aluminium Co. Ltd, Chalfont Park, Bucks. We thank Dr Jill Curtis for her helpful advice. REFERENCES BADENOCH-JONES P., TURK J.L. & PARKER D. (1978) The effects of some aluminium and zirconium compounds on

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guinea-pig peritoneal macrophages and skin fibroblasts in culture. J. Path. 124, 51. BITTER-SUERMANN D., HADDING U., SCHORLEMMER H.U., LIMBERT H., DIERICH M. & DUKOR P. (1975) Activation by some T-independent antigens and B cell mitogens of the alternative pathway of the complement system. J. Immunol. 115,425. BLACK M.N. & EPSTEIN W.L. (1974) Formation of multinucleate giant cells in organized epithelioid cell granulomas. Am. J. Path. 74, 263. DEPASSE J. & LEONIS J. (1974) Toxicity of quartz and kaolin towards membranes: effect of lysozyme. Archs int. Physiol. Biochim. 82, 980. DEUTSCH D.G. & MERTZ E.T. (1970) Plasminogen: purification from human plasma by affinity chromatography. Science, 170, 1095. FiNE D.P., MARNEY JR. S.R., COLLEY D.G., SERGENT J.S. & DEs PREZ R.M. (1972) C3 shunt activation in human serum chelated with EDTA. J. Immunol. 109, 807. HUDSON L. & HAY F.C. (1976) Practical Immunology p. 139. Blackwell Scientific Publications, Oxford. KABAT E.A. & MAYER M.M. (1961) Experimental Immunochemistry 2nd ed. p. 288. Charles C. Thomas, Springfield, Illinois. KLEINE I., POPPE B. & VOGT W. (1970) Functional identity of AT preparations obtained from different sources and by different activation procedures. 1. Pharmacological experiments. Europ. J. Pharm. 10, 398. KOLB W.P., LANG J.H. & LASSER E.C. (1978) Non-immunological complement activation in normal human serum induced by radiographic contrast media. J. Immunol. 121, 1232. LACHMANN P.H. (1975) Complement. In: Clinical aspects of immunology. (Ed. by P.G.H. Gell, R.R.A. Coombs and P.J. Lachmann) p.333. Blackwell Scientific Publications, Oxford. LASSER E.C., FARR R.S., FUJIMAGARI T. & TRIPP W.N. (1962) The significance of protein binding of contrast media in Roentgen diagnosis. Am. J. Roentgenol. 87, 338. LORD G.H. & WILSON, J.E. (1968) Foreign body granuloma in a rhesus monkey. J. Am. vet. med. Ass. 153, 910. LOWRY O.H., ROSENBROUGH N.J., FARR A.L. & RANDALL R.J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. LYNCH K.M. & MCIVER F.A. (1954) Pneumoconiosis from exposure to kaolin dust. Am. J. Path. 30, 1117. MAYER M.M. (1961) Complement and complement fixation. In: Experimental Immunochemistry 2nd edn (Ed. by E. A. Kabat & M. M. Mayer), p. 133. Charles C. Thomas, Springfield, Illinios.

MORGAN A., HOLMES, A. & TALBOT R.J. (1977) The haemolytic activity of some fibrous amphiboles and its relation to their specific surface areas. Ann. ocup. Hyg. 20, 39. OGSTON D., OGSTON C.M., RATNOFF O.D. & FORBES C.D. (1969) Studies on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation ofthe Hageman factor and additional cofactors. J. clin. Invest. 48, 1786. PERRY K.M.A. (1963) In: Occupational Lung Diseases: Chest Diseases, Vol 1 (Ed. by K.M.A. Perry & T.H. Sellors), p.517. Butterworth, London. POLLEY M.J. & NACHMANN R.L. (1975) Ultrastructural lesions on the surface of platelets associated with either blood coagulation or with antibody-mediated immune injury. J. exp. Med. 141, 1261. PROOM H. (1943) The preparation of precipiating antisera for the identification of animal species. J. Path. Bact. 55,419. RATNOFF O.D. & NAFF G.G. (1967) The conversion of C'l s to C'2 esterase by plasmin and trypsin. J. exp. Med. 125, 337. REED R.E., VALERIO M.G., ULLAND B.M., VALERIO D.A. & STOKKEY J.L. (1970) Mediastinal subcutaneous cervical granulomas produced by faulty esophageal intubation of kaolin mixture in Macaques. Lab. Animal Care, 20, 720. ROCHA E., SILVA M. & ROTHSCHILD A.M. (1956) Experimental design for bioassay of a material inducing strong tachyphylactic effect (anaphylatoxin). Br. J. Pharm. 11, 252. SCHORLEMMER H.U., BITTER-SUERMANN D. & ALLISON A.C. (1977) Complement activation by the alternative pathway and macrophage enzyme secretion in the pathogenesis of chronic inflammation. Immunology, 32, 929. SHELLEY W.B. & HURLEY H.J. (1958) The allergic origin of zirconium deodorant graiiulomas. Br. J. Derm. 70, 75. TILL G., ROTHER U. & GEMSA D. (1978) Activation of complement by radiographic contrast media: generation of chemotactic and anaphylatoxin activities. Int. Archs Allergy appl. Immun. 56, 543. TURK J.L. & PARKER D. (1977a) Granuloma formation in normal guinea pigs injected intradermally with aluminium and zirconium compounds. J. invest. Derm. 68, 336. TURK J.L. & PARKER D. (1977b) Sensitization with Cr, Ni and Zr salts and allergic type granuloma formation in the guinea pig. J. invest. Derm. 68, 341. TURK J.L., BADENOCH-JONES P. & PARKER D. (1978) Ultrastructural observations on epithelioid cell granulomas induced by zirconium in the guinea pig. J. Path. 124, 45. WARD P.A. (1967) A plasmin-split fragment of C'3 as a new chemotactic factor. J. exp. med. 126, 189.

Complement activation by aluminium and zirconium compounds.

Immunology 1979 37 881 Complement activation by aluminium and zirconium compounds V. D. RAMANATHAN, P. BADENOCH-JONES & J. L. TURK Department of Pat...
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