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Low molecular weight IgM in healthy adults: Influence of HLA
ELSF\‘IER
Low Molecular Weight IgM in Healthy Adults: Influence of HLA Huji Xu and Peter J. Roberts-Thomson ABSTRACT: Circulating LMW IgM was detected in a group of 100 healthy adults, but in very low or trace concentrations. A significant correlation was observed between total IgM and LMW IgM (r = 0.81, p < O.Ol), which suggests that the presence of LMW IgM may be due to the “overspill” of LMW IgM during IgM synthesis and secretion. Significantly higher levels of LMW IgM
ABBREVIATIONS HIV human immunodeficiency HMW high molecular weight LMW low molecular weight
and total IgM were found in females as compared with males. Furthermore, a weak association between Al, B8, DR3 and elevated IgM was also found. However the pre0 Americise significance of this association is unclear. can Society for Histocompatibility and Immunogenetics, 1996. Human Immunology 51, 55-59 (1996)
HLA MHC RA
virus
human leucocyte antigen major histocompatibility complex rheumatoid arthritis
INTRODUCTION Low Molecular Weight (LMW) IgM, the monomeric subunit of high molecular weight or pentameric IgM, is a naturally occurring monomeric form of circulating IgM 11, 21. Currently there are several techniques available for the detection and quantification of LMW IgM including immunodiffusion 131, immunoelectrophoresis 141, PHA selection electrophoresis 151, radial immunodiffusion 161, filtration chromatography 127, and immunoblot technique 171. Using less sensitive methods such as immunodiffusion technique or filtration chromatography, circulating LMW IgM has been detected rarely or only in trace quantities in the sera of healthy adults, but it is frequently found in high concentrations in certain disorders 181. However, using a sensitive enhanced chemiluminescence detection system combined with a modified immunoblot technique, LMW IgM has been found in the sera of all healthy adults that have been tested, although in low levels or in trace quantities 171.
From the Department of Clinical Immunology, Flindm Medical Centre. Flindws University of South Australia, Bedford Park, South Australia, 5042. Correspondence to: AIProf: Peter Roberts-Thomson, Department of Clinical Immunology. Flinders Medical Centre, Bedford Park, SA 5042, Australia. Received January 24, 1996: accepted June 24, 1996. Human Immunology 51, 55-59 (1996) 0 American Society for Histocompatibility
and Immunogenetics,
1996
Uncertainty exists regarding the significance of low levels or trace quantities of LMW IgM in normal adult serum. In diseased individuals, however, high levels of LMW IgM have been reported to be significantly associated with the levels of total IgM, rheumatoid factor, and with circulating immune complexes in rheumatoid arthritis (RA), primary biliary cirrhosis, infective endocarditis, and selective IgA deficiency, etc. 12, 9-131. Furthermore, previous studies have shown a strong association between the levels of LMW IgM and the presence of rheumatoid vasculitis and the severity of disease in RA and in systemic lupus erythematosus 12, 8, 13}. Its close association with indices of disease severity or activity suggests that LMW IgM may play an important role in the immunopathogenesis of those diseases {Sl. In many of these diseases the role of genetic influences in the pathogenesis has been clearly established. For example, it has been demonstrated that a link between RA and HLA-D class II major histocompatibility molecular complex, notably DR, and DR, exists 114, 151, although the association is not absolute. Moreover, RA affects approximately 1-3 percent of the population, with a female to male ratio of 3:l. More recently, Simmonds and his colleagues 1161 have observed that a subgroup of individuals with human immunodeficiency virus (HIV) infection who had high IgM levels in the initial or early phase of 0198~8859/96/$15.00 PII SO198-8859(96)00152-S
56
H. Xu and P. J. Roberts-Thomson
their infection had a worse prognosis and progressed to the related acquired immunodeficiency syndrome illness and death more quickly than those who had low or normal IgM levels. Furthermore, in that particular group the HLA haplotype Al, BS, and DR3 was weakly associated with an increased risk of seroconversion on exposure to the virus 1171. Thus these findings suggest that genetic determinations are important in certain aspects in the above mentioned diseases. The two-fold aim of this study is to investigate the occurrence of circulating LMW JgM and IgM in healthy adults and to investigate the genetic influence on IgM levels. We have investigated the gender influence on the levels of total IgM and LMW IgM and have addressed the question as to whether there are any associations between IgM levels and HLA phenotypes, particularly Al, B8, and DR3.
MATERIALS Normal
AND
Adult
METHODS
of Total
Serum IgM was measured mans ICS). The interassay measurement was 3.8%. Detection
Total
IgM and LMW
IgM in Normal
Sera
Sera from 100 normal blood donors were measured by laser nephelometry for total IgM. The IgM levels varied from 0.46 g/L to 4.03 g/L, with a mean 1.51 g/L f 0.11 (Fig. 1). By means of a modified immunoblot technique combined with an enhanced chemiluminescence detection system, LMW IgM was also detected in all these sera (Fig. 2). The percentage of LMW IgM in normal sera was all less than 15% of total IgM with the total level of 0.04 g/L to 0.9 g/L and mean 0.27 g/L i 0.03. There was a significant correlation between the levels of total IgM and LMW IgM (r = 0.807, p < 0.01; Fig. 3).
of LMW
Total
IgM and LMW
in Female
and Male Donors
A comparison of circulating total IgM and LMW IgM in female and male blood donors is shown in Fig. 4. A significant higher level of total IgM in females was found FIGURE 1 The levels of total IgM and LMW IgM in the sera of the 100 blood donors.
IgM by laser nephelometry coefficient of variance
4.0
(Beckfor this
l* . .
a
IgM
LMW IgM was detected by an enhanced chemiluminescence detection system combined with a modified immunoblot technique [7]. In brief, serum and culture supernatant was separated on 3.6% SDS-PAGE and the separated serum proteins transferred to nitrocellulose; the IgM bands were developed with HRP-conjugated secondary antibody (Silenus Laboratory, Victoria); and the binding finally was detected by the enhanced chemiluminescence detection system (Amersham, UK). After immunoblotting, the blot was scanned using a Camag electrophoresis scanner; and the areas subtended by the LMW IgM peak were weighted and expressed in mg quantities. Statistical
RESULTS
Sera
One hundred normal adult sera were obtained from the South Australian Red Cross. There were 55 women and 45 men, ranging in age from 18 to 55 years. The phenotypes of class I and Class II antigens in these donors were determined by serological and hybridization methods as has been described in the routine tissue typing laboratory of the Red Cross [lS]. The sera were aliquoted into small volumes and stored frozen at -70°C. Measurement
nors for total IgM and LMW IgM was performed using the student’s t-test. The method of Fisher’s exact test was used for the analysis of association between the levels of IgM and certain HLA phenotypes. Significance was accepted if p < 0.05.
3.0
.
pn 2.0
1.0
Analysis
The linear correlation method was used for the analysis of any correlations between the levels of total IgM and LMW IgM. Comparison between female and male do-
0.0 Total IgM
LWM IgM
o
Male Donors
l
Female Donors
Low Molecular Weight
1
2 3
4
57
IgM and HLA
5
6
7 8
9 10 11 1213
FIGURE 2 Immunoblot appearance of normal sera from healthy adults (lane 1-14). One distinct monomeric IgM band (2 x 10s D) can be seen in all sera in addition to the penta-
when 1.315 LMW with 0.01).
compared with male donors (1.698 g/L k 0.123 vs g/L + 0.095, p < O.OS). Similarly, higher levels of IgM were also found in female donors as compared males (0.34 g/L f 0.03 vs 0.23 g/L + 0.02, p <
FIGURE 3 The correlation between total IgM and LMW IgM in 100 normal sera. A significant correlation between them was noted (r = 0.807, p < 0.01). 1.0 y = - 4.4424e-2+ 0.21360x RA2 = 0.307
merit band (1 x 10” D). Lane 15 is a positive control serum containing large quantities of monomeric and oligomeric IgM.
Total IgM and LMW IgM associated Phenotypes
with HLA
The normal range of IgM in adults is 0.5 to 2.5 g/L, as measured by laser nephelometry. As such there were 12 blood donors in this study group having high levels of IgM (Table 1). Of these 12 donors, 3 bore the Al B8 FIGURE 4 Total IgM and LMW IgM in female and male donors. Data is expressed as a mean + S.E.M. from 55 female and 45 male blood donors. Gender effect was determined by p-values calculated using the Student t test.
21
0.8 -
1415
peo.05
0.6 -
H female male
q
$0l-
0-r Total IgM g/L
IgM
LMW IgM
58
H. Xu and P. J. Roberts-Thomson
TABLE
1
The HLA phenotypes donors
in high IgM blood
I.D.
HLA Phenotypes
A3 B3 D2 D6 G6 H4
A3,25; B49,B35; DR13; DRW52,53; DQWl,S A2,A29; B44; DR13,DRll; DRW52; DQWl,3 Al,A25; B44,B37; DRl,DR4; DRW53; DQW1,3 Al,A3; B7,B8; DR3,DR4; DRW52,53; DQW2,3 A2,A24; B8,B51; DR2,DRll; DRW52; DQW1,3 Al l,A24; B27,Bbl; DR4,DR8; DRW52,53; DQWS Al,A2; B44,B8; DR3,DR4; DRW52,53; DQW2,3 A28,A31; B8; DR3; DRW52; DQW2 Al,A31; B8,B57; DR3; DRW52; DQW2 A2,A3; B44,B7; DR15,DR4; DRW53; DQW1,3 A2,All; BSl,B35; DRl,DR7; DRW53; DQWl,Z A2,A32; BS,B35; DR3,DR14; DRW52; DQW1,2
I3 14
f
‘l
K2 K4
IgM (g/L) 4.03 3.43 2.51 3.93 2.71 4.00 2.47 3.83 2.45 3.46 2.99 2.66
DR3 haplotype. Of the 100 blood donors in the study group, 13 individuals bore the Al B8 DR3 haplotype. Thus the frequency of high IgM donors with this HLA haplotype was 25%, while for others having normal IgM with this haplotype the frequency was 11.3% (10 out of 88). Accordingly, a relative risk for high levels of IgM with Al B8 DR3 was 2.6.
DISCUSSION Using the enhanced chemiluminescence detection system combined with a modified immunoblotting technique, it has been shown, for the first time, that circulating LMW IgM occurred in the sera of all healthy adults (Fig. 2). However, LMW IgM in these sera was present in low levels or only in trace quantities. This finding suggests that the previous failure to detect LMW IgM in normal sera was most likely due to the levels of LMW IgM being lower than the threshold sensitivities of the applied assays since the sensitivity of the chemiluminescence detection system is considerably greater than the earlier techniques 171. The significance of low levels of LMW IgM in the normal sera is uncertain. To explore this question, we compared LMW IgM levels with total concentrations of circulating IgM. A significant correlation was found (r = 0.807, p < 0.01) (Fig. 3). Since total IgM levels measured by laser nephelometry in these 100 samples represented a normal range distribution (except 12 who had high levels of IgM) (Fig. 1; Table l), the correlation between the levels of total IgM and LMW IgM may simply indicate that LMW IgM is a “normal component” in serum in healthy adults. It is hypothesized that its presence may be attributed to an “overspill” during pentameric IgM synthesis and secretion 1191. Linkage of disease susceptibility to HLA has been already shown in certain autoimmune diseases. Of these
diseases, some (such as RA) have high levels of circulating LMW IgM. Are there any associations between the levels of IgM (and LMW IgM) with HLA phenotypes in healthy and/or diseased individuals? This question has not been addressed previously. From this study, it was observed that female blood donors exhibited significantly higher levels of IgM than did male blood donors (Fig. 4). Similarly, significantly high levels of LMW IgM were also found in the female group (Fig. 4). These findings indicate that gender is an important factor in determining levels of IgM. In order to further analyze the association between IgM levels and HLA phenotypes, the blood donors were divided into two subgroups, according to their IgM levels. There were 12 blood donors who had high levels of IgM, while the remaining 88 blood donors were normal. In analysis of the association, the level between a particular disease and an allele at one of the MHC loci is defined in terms of relative risk. In general, a relative risk greater than 1 indicates an association between the disease and the antigen in question; and the greater the relative risk value, the greater this association. However, the statistical significance of an association depends upon both the relative risk and the number of individuals studied 1201. In the present study, a weak association between Al B8 DR3 haplotype and an elevated IgM was found, with a relative risk of high levels of IgM with Al B8 DR3 being 2.6. In fact, a research group in Edinburgh has also reported that the course of HIVassociated disease was related to at least two patient characteristics recognizable before exposure to the virusnamely, HLA type (Al B8 DR3) and total plasma IgM concentration [lb, 171. Nevertheless, the mechanism by which HLA alleles are associated with disease susceptibility (or, as in this study, specifically with elevated IgM) is unclear and forms the basis of many current investigations.
ACKNOWLEDGMENTS
We thank Professor James McCluskey at the Australian Cross for providing the normal blood samples.
Red
REFERENCES Harisdangkul V, McDougal JS, Knapp M, Christian CL: Naturally occurring low molecular weight IgM in patients with rheumatoid arthritis, systemic lupus erythematosus and macroglobulinemia. I. Purification and immunologic studies. J Immunol 11~216, 1975. Roberts-Thomson PJ, Wernick RM, Ziff M: Low molecular weight IgM in rheumatoid arthritis and other rheumatic diseases. Arthritis Rheum 24:795, 1981. Strobo JD, Tomasi
TB Jr: A low molecular
weight
im-
Low Molecular
Weight
59
IgM and HLA
munoglobulin antigenically Invest 46:1337, 1967.
related
to 19s IgM. J Clin
4. Salmon A: Molecular heterogeneity of immunoglobulin-M (rM-Globulin). J Immunol 102:496, 1969. 5. Spengler GA, Weber RM: PHA selection electrophoresis-A screening method for monomeric IgM. J Immunol Methods 32:7 1, 1980. 6. Coughlan RJ: Single step radial immunodiffusion (RID) method for the quantitation of monomeric IgM. J Clin Lab Immunol 15:51, 1984. K, McNeilage J, Gordon TP, 7. Xu HJ, Umapathysivam Roberts-Thomson PJ: An enhanced chemiluminescence detection system combined with a modified immunoblot technique for the detection of low molecular weight IgM in sera from healthy adults and neonates. J Immunol Methods 146:241, 1992. PJ: 8. Xu HJ, Roberts-Thomson weight IgM-A disease marker immunodeficient and B cell ders. Disease Markers 10:115,
Circulating low molecular in autoimmune, infective, lymphoproliferative disor1992.
PJ, Shearman DJC: 12. Kwitko A, Roberts-Thomson molecular weight IgM in selective IgA deficiency. Exp Immunol 50:198, 1982.
Low Clin
PJ: Low molecular 13. Xu HJ, Geddes R, Roberts-Thomson weight IgM and the role of CD5 B cells in rheumatoid arthritis. Ann Rheum Dis 53:383, 1994. 14. Thomsen M, Morling N, Snorrason E, et al.: HLA-DW4 and rheumatoid arthritis. 13:56, 1979. 15. Khan MA, Kushner I, Braun WE, et al.: Clinical and HLA studies in multiple case families with rheumatoid arthritis. Tissues and Antigen 18:136, 1981. P, Beatson D, Cuthbert RJG, Watson H, 16. Simmonds Reynolds B, Peutherer JF, Parry JV, Ludlam CA, Steel CM: Determinants of HIV disease progression: six-year longitudinal study in the Edinburgh haemophilia/HIV cohort. Lancet 338:1159, 1991. 17. Steel CM, Ludlam CA, Beatson D, Peutherer JF, Cuthbert RJG, Simmonds P, Morrison H, Jones M: HLA haplotype Al B8 DR3 as a risk factor for HIV-related disease. Lancet May:1185, 1988.
V, Barnes TY, Sogcharoen S, Pennebaker 9. Harisdangkul JB: Clinical significance of low molecular weight IgM in patients with systemic lupus erythematosus. J Rheumatol 11:63, 1984.
18. Tay GK, Witt CS, Christiansen FT, Charron D, Baker D, Herrmann R, Smith LK, Diepeveen D, Mallal S, McCluskey J, et al.: Matching for MHC haplotypes results in improved survival following unrelated bone marrow transplantation. Bone Marrow Transplant 15:381, 1995.
F, DeViliers D, Thomas HC, 10. Fakunle YM, Aranguibel Slerlock S: Monomeric (7s) IgM in chronic liver diseases. Clin Exp Immunol 38204, 1979.
PJ: Appearance of 19. Koh LY, Jones DN, Roberts-Thomson low molecular weight IgM during course of infective endocarditis. Clin Exp Immunol 64:471, 1986.
11. Koh LY, Jones DN, Roberts-Thomson PJ: Appearance of low molecular weight IgM during course of infective endocarditis. Clin Exp Immunol 64:471, 1986.
20. Hansen TH, Sachs DH: The major histocompatibility complex. In Paul WE (ed.): Fundamental Immunology, 2d ed., Raven Press Ltd., New York, p 445, 1989.
Low Molecular Weight IgM in Healthy Adults: Influence of HLA Huji Xu and Peter J. Roberts-Thomson ABSTRACT: Circulating LMW IgM was detected in a group of 100 healthy adults, but in very low or trace concentrations. A significant correlation was observed between total IgM and LMW IgM (r = 0.81, p < O.Ol), which suggests that the presence of LMW IgM may be due to the “overspill” of LMW IgM during IgM synthesis and secretion. Significantly higher levels of LMW IgM
ABBREVIATIONS HIV human immunodeficiency HMW high molecular weight LMW low molecular weight
and total IgM were found in females as compared with males. Furthermore, a weak association between Al, B8, DR3 and elevated IgM was also found. However the pre0 Americise significance of this association is unclear. can Society for Histocompatibility and Immunogenetics, 1996. Human Immunology 51, 55-59 (1996)
HLA MHC RA
virus
human leucocyte antigen major histocompatibility complex rheumatoid arthritis
INTRODUCTION Low Molecular Weight (LMW) IgM, the monomeric subunit of high molecular weight or pentameric IgM, is a naturally occurring monomeric form of circulating IgM 11, 21. Currently there are several techniques available for the detection and quantification of LMW IgM including immunodiffusion 131, immunoelectrophoresis 141, PHA selection electrophoresis 151, radial immunodiffusion 161, filtration chromatography 127, and immunoblot technique 171. Using less sensitive methods such as immunodiffusion technique or filtration chromatography, circulating LMW IgM has been detected rarely or only in trace quantities in the sera of healthy adults, but it is frequently found in high concentrations in certain disorders 181. However, using a sensitive enhanced chemiluminescence detection system combined with a modified immunoblot technique, LMW IgM has been found in the sera of all healthy adults that have been tested, although in low levels or in trace quantities 171.
From the Department of Clinical Immunology, Flindm Medical Centre. Flindws University of South Australia, Bedford Park, South Australia, 5042. Correspondence to: AIProf: Peter Roberts-Thomson, Department of Clinical Immunology. Flinders Medical Centre, Bedford Park, SA 5042, Australia. Received January 24, 1996: accepted June 24, 1996. Human Immunology 51, 55-59 (1996) 0 American Society for Histocompatibility
and Immunogenetics,
1996
Uncertainty exists regarding the significance of low levels or trace quantities of LMW IgM in normal adult serum. In diseased individuals, however, high levels of LMW IgM have been reported to be significantly associated with the levels of total IgM, rheumatoid factor, and with circulating immune complexes in rheumatoid arthritis (RA), primary biliary cirrhosis, infective endocarditis, and selective IgA deficiency, etc. 12, 9-131. Furthermore, previous studies have shown a strong association between the levels of LMW IgM and the presence of rheumatoid vasculitis and the severity of disease in RA and in systemic lupus erythematosus 12, 8, 13}. Its close association with indices of disease severity or activity suggests that LMW IgM may play an important role in the immunopathogenesis of those diseases {Sl. In many of these diseases the role of genetic influences in the pathogenesis has been clearly established. For example, it has been demonstrated that a link between RA and HLA-D class II major histocompatibility molecular complex, notably DR, and DR, exists 114, 151, although the association is not absolute. Moreover, RA affects approximately 1-3 percent of the population, with a female to male ratio of 3:l. More recently, Simmonds and his colleagues 1161 have observed that a subgroup of individuals with human immunodeficiency virus (HIV) infection who had high IgM levels in the initial or early phase of 0198~8859/96/$15.00 PII SO198-8859(96)00152-S
56
H. Xu and P. J. Roberts-Thomson
their infection had a worse prognosis and progressed to the related acquired immunodeficiency syndrome illness and death more quickly than those who had low or normal IgM levels. Furthermore, in that particular group the HLA haplotype Al, BS, and DR3 was weakly associated with an increased risk of seroconversion on exposure to the virus 1171. Thus these findings suggest that genetic determinations are important in certain aspects in the above mentioned diseases. The two-fold aim of this study is to investigate the occurrence of circulating LMW JgM and IgM in healthy adults and to investigate the genetic influence on IgM levels. We have investigated the gender influence on the levels of total IgM and LMW IgM and have addressed the question as to whether there are any associations between IgM levels and HLA phenotypes, particularly Al, B8, and DR3.
MATERIALS Normal
AND
Adult
METHODS
of Total
Serum IgM was measured mans ICS). The interassay measurement was 3.8%. Detection
Total
IgM and LMW
IgM in Normal
Sera
Sera from 100 normal blood donors were measured by laser nephelometry for total IgM. The IgM levels varied from 0.46 g/L to 4.03 g/L, with a mean 1.51 g/L f 0.11 (Fig. 1). By means of a modified immunoblot technique combined with an enhanced chemiluminescence detection system, LMW IgM was also detected in all these sera (Fig. 2). The percentage of LMW IgM in normal sera was all less than 15% of total IgM with the total level of 0.04 g/L to 0.9 g/L and mean 0.27 g/L i 0.03. There was a significant correlation between the levels of total IgM and LMW IgM (r = 0.807, p < 0.01; Fig. 3).
of LMW
Total
IgM and LMW
in Female
and Male Donors
A comparison of circulating total IgM and LMW IgM in female and male blood donors is shown in Fig. 4. A significant higher level of total IgM in females was found FIGURE 1 The levels of total IgM and LMW IgM in the sera of the 100 blood donors.
IgM by laser nephelometry coefficient of variance
4.0
(Beckfor this
l* . .
a
IgM
LMW IgM was detected by an enhanced chemiluminescence detection system combined with a modified immunoblot technique [7]. In brief, serum and culture supernatant was separated on 3.6% SDS-PAGE and the separated serum proteins transferred to nitrocellulose; the IgM bands were developed with HRP-conjugated secondary antibody (Silenus Laboratory, Victoria); and the binding finally was detected by the enhanced chemiluminescence detection system (Amersham, UK). After immunoblotting, the blot was scanned using a Camag electrophoresis scanner; and the areas subtended by the LMW IgM peak were weighted and expressed in mg quantities. Statistical
RESULTS
Sera
One hundred normal adult sera were obtained from the South Australian Red Cross. There were 55 women and 45 men, ranging in age from 18 to 55 years. The phenotypes of class I and Class II antigens in these donors were determined by serological and hybridization methods as has been described in the routine tissue typing laboratory of the Red Cross [lS]. The sera were aliquoted into small volumes and stored frozen at -70°C. Measurement
nors for total IgM and LMW IgM was performed using the student’s t-test. The method of Fisher’s exact test was used for the analysis of association between the levels of IgM and certain HLA phenotypes. Significance was accepted if p < 0.05.
3.0
.
pn 2.0
1.0
Analysis
The linear correlation method was used for the analysis of any correlations between the levels of total IgM and LMW IgM. Comparison between female and male do-
0.0 Total IgM
LWM IgM
o
Male Donors
l
Female Donors
Low Molecular Weight
1
2 3
4
57
IgM and HLA
5
6
7 8
9 10 11 1213
FIGURE 2 Immunoblot appearance of normal sera from healthy adults (lane 1-14). One distinct monomeric IgM band (2 x 10s D) can be seen in all sera in addition to the penta-
when 1.315 LMW with 0.01).
compared with male donors (1.698 g/L k 0.123 vs g/L + 0.095, p < O.OS). Similarly, higher levels of IgM were also found in female donors as compared males (0.34 g/L f 0.03 vs 0.23 g/L + 0.02, p <
FIGURE 3 The correlation between total IgM and LMW IgM in 100 normal sera. A significant correlation between them was noted (r = 0.807, p < 0.01). 1.0 y = - 4.4424e-2+ 0.21360x RA2 = 0.307
merit band (1 x 10” D). Lane 15 is a positive control serum containing large quantities of monomeric and oligomeric IgM.
Total IgM and LMW IgM associated Phenotypes
with HLA
The normal range of IgM in adults is 0.5 to 2.5 g/L, as measured by laser nephelometry. As such there were 12 blood donors in this study group having high levels of IgM (Table 1). Of these 12 donors, 3 bore the Al B8 FIGURE 4 Total IgM and LMW IgM in female and male donors. Data is expressed as a mean + S.E.M. from 55 female and 45 male blood donors. Gender effect was determined by p-values calculated using the Student t test.
21
0.8 -
1415
peo.05
0.6 -
H female male
q
$0l-
0-r Total IgM g/L
IgM
LMW IgM
58
H. Xu and P. J. Roberts-Thomson
TABLE
1
The HLA phenotypes donors
in high IgM blood
I.D.
HLA Phenotypes
A3 B3 D2 D6 G6 H4
A3,25; B49,B35; DR13; DRW52,53; DQWl,S A2,A29; B44; DR13,DRll; DRW52; DQWl,3 Al,A25; B44,B37; DRl,DR4; DRW53; DQW1,3 Al,A3; B7,B8; DR3,DR4; DRW52,53; DQW2,3 A2,A24; B8,B51; DR2,DRll; DRW52; DQW1,3 Al l,A24; B27,Bbl; DR4,DR8; DRW52,53; DQWS Al,A2; B44,B8; DR3,DR4; DRW52,53; DQW2,3 A28,A31; B8; DR3; DRW52; DQW2 Al,A31; B8,B57; DR3; DRW52; DQW2 A2,A3; B44,B7; DR15,DR4; DRW53; DQW1,3 A2,All; BSl,B35; DRl,DR7; DRW53; DQWl,Z A2,A32; BS,B35; DR3,DR14; DRW52; DQW1,2
I3 14
f
‘l
K2 K4
IgM (g/L) 4.03 3.43 2.51 3.93 2.71 4.00 2.47 3.83 2.45 3.46 2.99 2.66
DR3 haplotype. Of the 100 blood donors in the study group, 13 individuals bore the Al B8 DR3 haplotype. Thus the frequency of high IgM donors with this HLA haplotype was 25%, while for others having normal IgM with this haplotype the frequency was 11.3% (10 out of 88). Accordingly, a relative risk for high levels of IgM with Al B8 DR3 was 2.6.
DISCUSSION Using the enhanced chemiluminescence detection system combined with a modified immunoblotting technique, it has been shown, for the first time, that circulating LMW IgM occurred in the sera of all healthy adults (Fig. 2). However, LMW IgM in these sera was present in low levels or only in trace quantities. This finding suggests that the previous failure to detect LMW IgM in normal sera was most likely due to the levels of LMW IgM being lower than the threshold sensitivities of the applied assays since the sensitivity of the chemiluminescence detection system is considerably greater than the earlier techniques 171. The significance of low levels of LMW IgM in the normal sera is uncertain. To explore this question, we compared LMW IgM levels with total concentrations of circulating IgM. A significant correlation was found (r = 0.807, p < 0.01) (Fig. 3). Since total IgM levels measured by laser nephelometry in these 100 samples represented a normal range distribution (except 12 who had high levels of IgM) (Fig. 1; Table l), the correlation between the levels of total IgM and LMW IgM may simply indicate that LMW IgM is a “normal component” in serum in healthy adults. It is hypothesized that its presence may be attributed to an “overspill” during pentameric IgM synthesis and secretion 1191. Linkage of disease susceptibility to HLA has been already shown in certain autoimmune diseases. Of these
diseases, some (such as RA) have high levels of circulating LMW IgM. Are there any associations between the levels of IgM (and LMW IgM) with HLA phenotypes in healthy and/or diseased individuals? This question has not been addressed previously. From this study, it was observed that female blood donors exhibited significantly higher levels of IgM than did male blood donors (Fig. 4). Similarly, significantly high levels of LMW IgM were also found in the female group (Fig. 4). These findings indicate that gender is an important factor in determining levels of IgM. In order to further analyze the association between IgM levels and HLA phenotypes, the blood donors were divided into two subgroups, according to their IgM levels. There were 12 blood donors who had high levels of IgM, while the remaining 88 blood donors were normal. In analysis of the association, the level between a particular disease and an allele at one of the MHC loci is defined in terms of relative risk. In general, a relative risk greater than 1 indicates an association between the disease and the antigen in question; and the greater the relative risk value, the greater this association. However, the statistical significance of an association depends upon both the relative risk and the number of individuals studied 1201. In the present study, a weak association between Al B8 DR3 haplotype and an elevated IgM was found, with a relative risk of high levels of IgM with Al B8 DR3 being 2.6. In fact, a research group in Edinburgh has also reported that the course of HIVassociated disease was related to at least two patient characteristics recognizable before exposure to the virusnamely, HLA type (Al B8 DR3) and total plasma IgM concentration [lb, 171. Nevertheless, the mechanism by which HLA alleles are associated with disease susceptibility (or, as in this study, specifically with elevated IgM) is unclear and forms the basis of many current investigations.
ACKNOWLEDGMENTS
We thank Professor James McCluskey at the Australian Cross for providing the normal blood samples.
Red
REFERENCES Harisdangkul V, McDougal JS, Knapp M, Christian CL: Naturally occurring low molecular weight IgM in patients with rheumatoid arthritis, systemic lupus erythematosus and macroglobulinemia. I. Purification and immunologic studies. J Immunol 11~216, 1975. Roberts-Thomson PJ, Wernick RM, Ziff M: Low molecular weight IgM in rheumatoid arthritis and other rheumatic diseases. Arthritis Rheum 24:795, 1981. Strobo JD, Tomasi
TB Jr: A low molecular
weight
im-
Low Molecular
Weight
59
IgM and HLA
munoglobulin antigenically Invest 46:1337, 1967.
related
to 19s IgM. J Clin
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