741 We thank the obstetric and midwifery staff at both hospitals for their cooperation; Dr Margaret Harper, Dr Stella Okonkwo, and Dr Francoise Shenfield for their assistance in obtaining blood-samples and following up the mothers; Mr Graham Johnson and Miss Mary Zambon, of the department of chemical pathology, for their help.
Requests for reprints should be addressed to T.E.O. REFERENCES D.
1. Lakdawala, R., Widdowson, E. M. Lancet, 1977, i, 167. 2. Lancet, 1977, i, 840. 3. Friesen, H. G., Hwang, P. A. Rev. Med. 1973, 24, 251. 4. McLaughlin, M., Fairney, A., Lester, E., Raggatt, P. R., Brown, D.
J., Wills,
M. R. Lancet, 1974, i, 536. 5. Turton, C. W. G., Stamp, T. C. B.,
Stanley, P., Maxwell, J. D. ibid. 1977, i, 222. 6. Belsey, R. E., De Luca, H. F., Potts, J. T. J. clin. Endocr. Metab. 1974, 38, 1046. 7. Haddad, J. G., Min, C., Walgate, J. in Vitamin D Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. (edited by A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigoleit, and D. von Herrath); p. 463. Berlin, 1977.
COMPLEXED
IgE IN ATOPY
JONATHAN BROSTOFF Department of Immunology, Middlesex Hospital Medical School, London W1 PHILIP
DENNIS R. STANWORTH JOHNS Department of Experimental Pathology, University of Birmingham, Birmingham
Evidence of circulating high-molecularweight forms of IgE was found in 16 patients with hayfever and/or atopic eczema. This finding of what appears to be complexed IgE in the serum of atopic patients may help to elucidate the ætiology of
Summary
atopy. è1lUpy,
Introduction ONE of the hallmarks of the patient with atopic eczema, asthma, and positive immediate weal-and-flare skin reactions is the very high serum-IgE concentration. These concentrations are often much higher than those of patients with eczema.! Moreover, skin lesions in atopic eczema do not have the histological appearance of a classic immediate hypersensitivity reaction. The picture is mixed with small round cell infiltration in the dermis predominating, and is more reminiscent of delayed
FRACTIOfiI
A
RU6ER
typical density gradient ultracentrifugation profile of serum from a patient with atopic eczema and asthma, showing distribution of IgE expressed as radioactive 1251 counts per frac-
tion. Normal sedimentation distribution of a radiolabelled IgE myeloma protein, ultracentrifuged under identical conditions, has been superimposed for comparison.
hayfever alone. A further 4 sera from hay-fever patients were kindly supplied by Dr John Morrison Smith of Birmingham Chest Hospital. Sera.-Blood from patients was allowed to clot at room temperature, was centrifuged, and was then stored at -20°C. Four sera were assayed for complexes on the same day as the blood was taken to avoid any possible aggregation by freezing. Ultracentrifugation.-Serum samples (0.2 ml) were centrifuged on an isokinetic sucrose density gradient with a top sucrose concentration of 10% (w/v). The gradients were centrifuged for eighteen hours at 25 000-35 000 r.p.m. at 20°C in a Beckman SW 41 Ti rotor in a Beckman L2-65 preparative ultracentrifuge.4 The gradient was monitored by ultraviolet light absorption (280 nm) by means of upward displacement by a 60% sucrose solution saturated with tyrosine in a Gilford 2480 density gradient scanner attached to a Gilford 2000 multiple sample absorbance recorder. The eluate was separated into 5-drop fractions with an LKB’Ultrorac’ Each fraction was assayed for total IgE content by the RIST (radioimmunosorbent SIZE DISTRIBUTION OF
IgE-CONTAINING COMPONENTS
DETERMINED BY ISOKINETIC DENSITY GRADIENT
ULTRACENTRIFUGATION
hypersensitivity.2 Immune complexes can cause lesions resembling delayed-hypersensitivity skin reactions.3 As an explanation of the paradoxical finding of a high serum-IgE associated with a cell-mediated histological picture, we wondered whether immune complexes containing IgE might
be found in the
serum
of
patients
with
atopic
eczema.
We used refined ultracentrifugation procedures to look for complexed IgE in sera from patients with atopic eczema as well as from patients with hayfever alone (as controls). We made no a priori judgement as to the other components of the complex, which could include antigen
(allergen), antiglobulin (i.e., IgG anti-IgE), or both. Methods Patients
selected from the Allergy Clinic at the Middlesex Hospital, and included 10 patients who had atopic eczema complicated by asthma and/or hayfever and 2 with were
Mean monomer sedimentation coefficient (So.w)=7.97:t0.35 (11 determinations); corresponds to a molecular weight of 210 000. Mean sedimentation coefficient (S,.w’’ of 125I-IgE (N.D.)=7. 71 :to.32 (17 determinations); corresponds to a molecular weight of 207 000.
742
test) method. By reference
to the ultraviolet trace the position of the IgE-containing components could be determined,. and from this the sedimentation coefficient could be estimated. We used an empirical relation between sedimentation coefficient and molecular weightS to estimate the molecular weight of the IgE-containing components. A similar ultracentrifugal technique was applied to the measurement of 1251-labelled IgE in which the fractions were counted directly in a gamma counter. IgE assay.-The total IgE content of ultracentrifugation fractions and starting sera were determined by the standard RIST procedure with sheep antibody directed against the IgE myeloma protein N.D.6
Results
patients studied, 15 contained complexed IgE-i.e., IgE with a sedimentation coefficient greater than 8S was detectable. An IgE myeloma (125 I-labelled) protein studied at the same time by a similar technique showed a sharp peak in the region of 8S. A typical ultracentrifugal scan and the distribution of IgE in the corresponding fractions is shown in the accompanying figure, on which has been superimposed a trace of the distribution of radioactivity in a radiolabelled (1251) myeloma IgE preparation ultracentrifuged Of the
sera
of the 16
under identical conditions. Of the group of 16 patients investigated, 10 had atopic eczema and all of these had high-molecular-weight IgE in their sera (see accompanying table). None of the 3 sera from patients with atopic eczema and low serumIgE concentrations (patients 7, 8 and 9) appeared to contain any monomeric IgE, all of the detectable immunoglobulin being in the polymeric form in these sera. Where complexed IgE was present as well as monomer-i.e., in 12 out of 13 of the remaining sera examined-it comprised about 50% of the total IgE (range
I ,
20-70%). Discussion
.
By application of definitive ultracentrifuge analysis we found evidence of complexed IgE in the serum not only of patients with atopic eczema, but also of those with hayfever alone. These complexed forms of IgE do not seem to be formed by aggregation of the immunoglobulin on freezing, since 4 fresh sera also contained complexed IgE. This raised the question of whether these findings are directly related to the disease or whether they reflect a more basic immunological defect. This question can be considered from several points of view. A small number of atopic individuals improve when the offending allergen is removed from their diet-e.g., exclusion of cow’s milk from the diet of some patients with atopic eczema. In this small group, the antigen (possibly 3-lactoglobulin) is the offending agent.On this basis, it is possible that we are observing IgE antigen complexes which are subsequently deposited in the skin of eczema patients thereby causing the lesions, as has been demonstrated in the model system of Spector and Heesom.3 This approach might lead ultimately to the identification of the offending allergen in these patients. Another possibility reflects the functions of rheumatoid factor. Both IgG and IgM antiglobulins (rheumatoid factors) seem to be produced in response to polymeric IgG or, more particularly, as a result of IgG complexed with antigen. Moreover, it has already been shown that IgG antiglobulin can be directed against IgE.7 It would seem possible, therefore, that the com-
demonstrated in our studies could be IgE combined with antiglobulin rather than IgE/allergen complex. Further studies are in progress to investigate this possibility, including ultracentrifuge analysis of sera from allergic patients under conditions known to dissociate antigen/antibody complexes. A third possible interpretation of our observations stems from the work of Soothill and Steward8 who have shown that some animals make poor-affinity antibody which leads to the formation of soluble immune complexes. The inference to be drawn from their studies, and our present findings of complexed IgE in the serum of both the eczematous and hayfever patients, is that atopic patients in general could be producers of pooraffinity IgE antibody. The recent findings of Godfrey9 tend to support this idea. However, complexes (22S) consisting of low-affinity rheumatoid factor (IgM antibody) and IgG become dissociated under similar densitygradient sedimentation conditions to those used in the present study, 10 and we are, therefore, presumably studying complexes containing antibody of much higher affinity. We feel that it is unlikely that atopic patients in general are genetically producers of poor-affinity IgE There is antibody, although this remains a possibility. also no strong HLA linkage with atopy."I Our preliminary findings of what appears to be complexed IgE in the serum of atopic patients raises many questions about the possible presence and nature of other components in the complex. Studies in this direction now in progress could throw further light on the aetiology of atopy. We thank Mrs. J. Wilson for assistance with the IgE estimations.
plexed IgE
The financial support of the Asthma Research Council, and the Arthritis and Rheumatism Council is gratefully acknowledged. J. B. is supported by the Medical Research Council.
Requests for reprints should be addressed to J.B. REFERENCES
1. Turner, M. W., Brostoff, J., Soothill, J. F. Unpublished. 2. Mihm, M. C., Soter, N. A., Dvorak, H., Austen, K. F. J. invest. Derm. 1976,
67, 305. Spector, W. G., Heesom, W. J. Path. Bact 1969, 98, 31. 4. Johns, P., Stanworth, D. R. J. Immun. Meth. 1976, 10, 231. 5. Stewart, G. A., Johns, P. ibid. p. 219. 6. McLaughlan, P., Stanworth, D. R. Lancet, 1975, 64. 7. Williams, R. C., Griffiths, R. W., Emmons, J. D., Field, R. C. J. clin. Invest. 1972, 51, 955. 8. Soothill, J. F., Steward, H. W. Clin. exp. Immun. 1971, 9, 193. 9. Godfrey, R. C. Personal communication. 10. Normansell, D. E., Stanworth, D. R. Immunology, 1966, 10, 527. 11. Turner, M. W., Brostoff, J., Wells, R. S., Stokes, C. R., Soothill, J. F. Clin exp. Immun 1977, 27, 43. 3.
FAILURE OF SYNGENEIC BONE-MARROW GRAFT WITHOUT PRECONDITIONING IN POST-HEPATITIS MARROW APLASIA THE ROYAL MARSDEN HOSPITAL BONE-MARROW TRANSPLANTATION TEAM
Royal Marsden Hospital, Sutton, Surrey A
patient with post-hepatitis bone-maraplasia had bone-marrow transfrom an identical twin. The first two transplants plants were only partially and temporarily successful, but after cyclophosphamide treatment bone-marrow function was
Summary
row
restored. WE report the
problems
of bone-marrow
transplan-
Requests for reprints should be addressed to T.E.O. REFERENCES D.
1. Lakdawala, R., Widdowson, E. M. Lancet, 1977, i, 167. 2. Lancet, 1977, i, 840. 3. Friesen, H. G., Hwang, P. A. Rev. Med. 1973, 24, 251. 4. McLaughlin, M., Fairney, A., Lester, E., Raggatt, P. R., Brown, D.
J., Wills,
M. R. Lancet, 1974, i, 536. 5. Turton, C. W. G., Stamp, T. C. B.,
Stanley, P., Maxwell, J. D. ibid. 1977, i, 222. 6. Belsey, R. E., De Luca, H. F., Potts, J. T. J. clin. Endocr. Metab. 1974, 38, 1046. 7. Haddad, J. G., Min, C., Walgate, J. in Vitamin D Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. (edited by A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigoleit, and D. von Herrath); p. 463. Berlin, 1977.
COMPLEXED
IgE IN ATOPY
JONATHAN BROSTOFF Department of Immunology, Middlesex Hospital Medical School, London W1 PHILIP
DENNIS R. STANWORTH JOHNS Department of Experimental Pathology, University of Birmingham, Birmingham
Evidence of circulating high-molecularweight forms of IgE was found in 16 patients with hayfever and/or atopic eczema. This finding of what appears to be complexed IgE in the serum of atopic patients may help to elucidate the ætiology of
Summary
atopy. è1lUpy,
Introduction ONE of the hallmarks of the patient with atopic eczema, asthma, and positive immediate weal-and-flare skin reactions is the very high serum-IgE concentration. These concentrations are often much higher than those of patients with eczema.! Moreover, skin lesions in atopic eczema do not have the histological appearance of a classic immediate hypersensitivity reaction. The picture is mixed with small round cell infiltration in the dermis predominating, and is more reminiscent of delayed
FRACTIOfiI
A
RU6ER
typical density gradient ultracentrifugation profile of serum from a patient with atopic eczema and asthma, showing distribution of IgE expressed as radioactive 1251 counts per frac-
tion. Normal sedimentation distribution of a radiolabelled IgE myeloma protein, ultracentrifuged under identical conditions, has been superimposed for comparison.
hayfever alone. A further 4 sera from hay-fever patients were kindly supplied by Dr John Morrison Smith of Birmingham Chest Hospital. Sera.-Blood from patients was allowed to clot at room temperature, was centrifuged, and was then stored at -20°C. Four sera were assayed for complexes on the same day as the blood was taken to avoid any possible aggregation by freezing. Ultracentrifugation.-Serum samples (0.2 ml) were centrifuged on an isokinetic sucrose density gradient with a top sucrose concentration of 10% (w/v). The gradients were centrifuged for eighteen hours at 25 000-35 000 r.p.m. at 20°C in a Beckman SW 41 Ti rotor in a Beckman L2-65 preparative ultracentrifuge.4 The gradient was monitored by ultraviolet light absorption (280 nm) by means of upward displacement by a 60% sucrose solution saturated with tyrosine in a Gilford 2480 density gradient scanner attached to a Gilford 2000 multiple sample absorbance recorder. The eluate was separated into 5-drop fractions with an LKB’Ultrorac’ Each fraction was assayed for total IgE content by the RIST (radioimmunosorbent SIZE DISTRIBUTION OF
IgE-CONTAINING COMPONENTS
DETERMINED BY ISOKINETIC DENSITY GRADIENT
ULTRACENTRIFUGATION
hypersensitivity.2 Immune complexes can cause lesions resembling delayed-hypersensitivity skin reactions.3 As an explanation of the paradoxical finding of a high serum-IgE associated with a cell-mediated histological picture, we wondered whether immune complexes containing IgE might
be found in the
serum
of
patients
with
atopic
eczema.
We used refined ultracentrifugation procedures to look for complexed IgE in sera from patients with atopic eczema as well as from patients with hayfever alone (as controls). We made no a priori judgement as to the other components of the complex, which could include antigen
(allergen), antiglobulin (i.e., IgG anti-IgE), or both. Methods Patients
selected from the Allergy Clinic at the Middlesex Hospital, and included 10 patients who had atopic eczema complicated by asthma and/or hayfever and 2 with were
Mean monomer sedimentation coefficient (So.w)=7.97:t0.35 (11 determinations); corresponds to a molecular weight of 210 000. Mean sedimentation coefficient (S,.w’’ of 125I-IgE (N.D.)=7. 71 :to.32 (17 determinations); corresponds to a molecular weight of 207 000.
742
test) method. By reference
to the ultraviolet trace the position of the IgE-containing components could be determined,. and from this the sedimentation coefficient could be estimated. We used an empirical relation between sedimentation coefficient and molecular weightS to estimate the molecular weight of the IgE-containing components. A similar ultracentrifugal technique was applied to the measurement of 1251-labelled IgE in which the fractions were counted directly in a gamma counter. IgE assay.-The total IgE content of ultracentrifugation fractions and starting sera were determined by the standard RIST procedure with sheep antibody directed against the IgE myeloma protein N.D.6
Results
patients studied, 15 contained complexed IgE-i.e., IgE with a sedimentation coefficient greater than 8S was detectable. An IgE myeloma (125 I-labelled) protein studied at the same time by a similar technique showed a sharp peak in the region of 8S. A typical ultracentrifugal scan and the distribution of IgE in the corresponding fractions is shown in the accompanying figure, on which has been superimposed a trace of the distribution of radioactivity in a radiolabelled (1251) myeloma IgE preparation ultracentrifuged Of the
sera
of the 16
under identical conditions. Of the group of 16 patients investigated, 10 had atopic eczema and all of these had high-molecular-weight IgE in their sera (see accompanying table). None of the 3 sera from patients with atopic eczema and low serumIgE concentrations (patients 7, 8 and 9) appeared to contain any monomeric IgE, all of the detectable immunoglobulin being in the polymeric form in these sera. Where complexed IgE was present as well as monomer-i.e., in 12 out of 13 of the remaining sera examined-it comprised about 50% of the total IgE (range
I ,
20-70%). Discussion
.
By application of definitive ultracentrifuge analysis we found evidence of complexed IgE in the serum not only of patients with atopic eczema, but also of those with hayfever alone. These complexed forms of IgE do not seem to be formed by aggregation of the immunoglobulin on freezing, since 4 fresh sera also contained complexed IgE. This raised the question of whether these findings are directly related to the disease or whether they reflect a more basic immunological defect. This question can be considered from several points of view. A small number of atopic individuals improve when the offending allergen is removed from their diet-e.g., exclusion of cow’s milk from the diet of some patients with atopic eczema. In this small group, the antigen (possibly 3-lactoglobulin) is the offending agent.On this basis, it is possible that we are observing IgE antigen complexes which are subsequently deposited in the skin of eczema patients thereby causing the lesions, as has been demonstrated in the model system of Spector and Heesom.3 This approach might lead ultimately to the identification of the offending allergen in these patients. Another possibility reflects the functions of rheumatoid factor. Both IgG and IgM antiglobulins (rheumatoid factors) seem to be produced in response to polymeric IgG or, more particularly, as a result of IgG complexed with antigen. Moreover, it has already been shown that IgG antiglobulin can be directed against IgE.7 It would seem possible, therefore, that the com-
demonstrated in our studies could be IgE combined with antiglobulin rather than IgE/allergen complex. Further studies are in progress to investigate this possibility, including ultracentrifuge analysis of sera from allergic patients under conditions known to dissociate antigen/antibody complexes. A third possible interpretation of our observations stems from the work of Soothill and Steward8 who have shown that some animals make poor-affinity antibody which leads to the formation of soluble immune complexes. The inference to be drawn from their studies, and our present findings of complexed IgE in the serum of both the eczematous and hayfever patients, is that atopic patients in general could be producers of pooraffinity IgE antibody. The recent findings of Godfrey9 tend to support this idea. However, complexes (22S) consisting of low-affinity rheumatoid factor (IgM antibody) and IgG become dissociated under similar densitygradient sedimentation conditions to those used in the present study, 10 and we are, therefore, presumably studying complexes containing antibody of much higher affinity. We feel that it is unlikely that atopic patients in general are genetically producers of poor-affinity IgE There is antibody, although this remains a possibility. also no strong HLA linkage with atopy."I Our preliminary findings of what appears to be complexed IgE in the serum of atopic patients raises many questions about the possible presence and nature of other components in the complex. Studies in this direction now in progress could throw further light on the aetiology of atopy. We thank Mrs. J. Wilson for assistance with the IgE estimations.
plexed IgE
The financial support of the Asthma Research Council, and the Arthritis and Rheumatism Council is gratefully acknowledged. J. B. is supported by the Medical Research Council.
Requests for reprints should be addressed to J.B. REFERENCES
1. Turner, M. W., Brostoff, J., Soothill, J. F. Unpublished. 2. Mihm, M. C., Soter, N. A., Dvorak, H., Austen, K. F. J. invest. Derm. 1976,
67, 305. Spector, W. G., Heesom, W. J. Path. Bact 1969, 98, 31. 4. Johns, P., Stanworth, D. R. J. Immun. Meth. 1976, 10, 231. 5. Stewart, G. A., Johns, P. ibid. p. 219. 6. McLaughlan, P., Stanworth, D. R. Lancet, 1975, 64. 7. Williams, R. C., Griffiths, R. W., Emmons, J. D., Field, R. C. J. clin. Invest. 1972, 51, 955. 8. Soothill, J. F., Steward, H. W. Clin. exp. Immun. 1971, 9, 193. 9. Godfrey, R. C. Personal communication. 10. Normansell, D. E., Stanworth, D. R. Immunology, 1966, 10, 527. 11. Turner, M. W., Brostoff, J., Wells, R. S., Stokes, C. R., Soothill, J. F. Clin exp. Immun 1977, 27, 43. 3.
FAILURE OF SYNGENEIC BONE-MARROW GRAFT WITHOUT PRECONDITIONING IN POST-HEPATITIS MARROW APLASIA THE ROYAL MARSDEN HOSPITAL BONE-MARROW TRANSPLANTATION TEAM
Royal Marsden Hospital, Sutton, Surrey A
patient with post-hepatitis bone-maraplasia had bone-marrow transfrom an identical twin. The first two transplants plants were only partially and temporarily successful, but after cyclophosphamide treatment bone-marrow function was
Summary
row
restored. WE report the
problems
of bone-marrow
transplan-
