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Elevation of serum β2-microglobulin levels during infectious mononucleosis
CLINICAL
IMMUNOLOGY
Elevation
AND
24, 55-62 (1982)
IMMUNOPATHOLOGY
of Serum &Microglobulin Infectious Mononucleosis
Levels
during
JEAN-PIERRELAMELIN,“,’ CLAUDE VINCENT,$ CLAUDE FONTAINE-LEGRAND,+ AND JEAN-PIERRE REVILLARDS Lahorntories of *Immunology und tvirology, Centre Lyon. and $Luhoratory of Immunology. ERA-CNRS
Nutional de la Recherche 782, H6pital E. Herriot.
Scienti$qw. Lyon, Franw
&Microglobulin (P2m), a potential marker of in vitro T-cell activation, was measured in the earliest available serum sample from 28 acute infectious mononucleosis (I.M.) patients, all free of any renal dysfunction. The serum fi2rn (S p2m) level was higher than that of matched-healthy controls in 21. No correlation was found with the time elapsed from clinical onset, the clinical symptoms, or the percentage and absolute number of large lymphocytes. The highest S fi2rn levels were observed in three I.M. patients with the largest adenopathies. In nine of the patients, three to four sequential bleedings were examined. The S /32m increased levels returned to normal within 30 days, along with the main biological parameters of I.M. (large pyroninophilic cells, IgM anti-VCA antibody, circulating immune complexes). However, no correlation could be established in individual sera among them. These findings, along with other reports, indicate that T-cell activation results in increased P2m release and elevated S P2m levels. Determination of S fl2rn may therefore provide a global estimate of T-cell activation in viva. not necessarily correlated with changes in lymphocyte subsets that are amenable to study in peripheral blood.
INTRODUCTION
The assessment of the stimulation of the immune system in viva by the detection of soluble substances released by lymphoid cells would be of great help in the evaluation of immune disorders in man. So far, this has been achieved only for the B-cell compartment, by measuring the level of serum immunoglobulins. Conversely, the degree of activation of the T compartment has mainly relied on the enumeration and functional in vitro evaluation of T cells collected from the peripheral blood. The limitation of this approach is illustrated by several situations in which a discrepancy between lymphoid tissue and peripheral blood lymphocytes has been documented (1). P,-Microglobulin (/32m), a 12,000 molecular weight protein, noncovalently linked to glycoproteins bearing class I histocompatibility antigens on almost all cell membranes but especially on lymphocytes, may be considered as a potential marker for evaluating T-cell activation. Bernier and Fanger (2) first demonstrated that /32m was mainly a product of T lymphocytes since it was produced by peripheral blood lymphocytes when stimulated by phytohemagglutinin but not by the pokeweed mitogen. More recently it I Address reprint requests to J.-P. Lamelin, Laboratoire Carrel, 69372 Lyon Cedex 2, France.
d’immunologie-CNRS,
Faculte Alexis
5.5
0090-1229/82/070055-08$01.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form resewed.
56
LAMELIN
ET
AL.
was shown that using mixed populations of T and B lymphocytes of known ratios, the amount of /32m released in the culture medium from activated T cells was much higher than that of their B counterpart. It was then concluded that P2m release reflected primarily T-cell activation (3). Infectious mononucleosis (I.M.) is a benign lymphoproliferative disorder resulting from the primary infection with the Epstein-Barr virus (EBV) (4). This disease is characterized by a proliferation and activation of a majority of T lymphocytes in response to the transformation by EBV of a small number of B lymphocytes (5,6). This disease was therefore considered as a good clinical model for assessing whether changes in serum P2m (S /32) levels may be observed in the context of this well-characterized T-cell activation (7, 8). MATERIALS
AND METHODS
1. Patients. Twenty-eight patients with biologically confirmed I.M. were entered in the present study. Twenty-one were males and seven were females. Due to a biased access to patients (referred to us from either a children or a military hospital) there were two age groups: the nine children ranged from 5 to 10 and the young adults from 17 to 24. Biological evidence for I.M. came from positive Monospot (3 cases) or Paul-Bunnell-Davidsohn reaction (22 cases). In 13 subjects, including the 3 patients not diagnosed by the above tests, anti-Epstein-Barr virus antibodies were titrated (9). The serological pattern characteristic of I.M. was present in all: presence of IgM antibodies against the EBV capsid antigen (VCA) in the absence of detectable response towards the EB nuclear antigen (EBNA). Overt clinical manifestations including adenopathy, splenomegaly, and liver involvement (biologically confirmed) were recorded retrospectively from the medical charts. None of the patients had any past history of renal disease. Blood urea was normal and proteinuria absent in all cases. 2. S /32m determination. S /32m was evaluated by a radioimmunoassay set up with our reagents (10). /32m purified from the urine of renal transplanted patients was iodinated (1251)by the chloramine-T method. High avidity anti-/32m serum was raised in rabbit. The complexes 1251P2m-anti-/32m formed in the presence or absence of the test serum were precipitated in liquid phase of a guinea pig antirabbit Ig serum. Actual values were obtained from a standard curve run in parallel. Nineteen patients were bled once. Thirteen of them had their white blood cells (WBC) counted (and the differential established) within less than 24 hr. Nine patients were bled sequentially, 2 weeks apart, three times, and, in two cases, four times. In this group of patients, the WBC differential count was determined on Ficoll-Isopaque separated cells. The time elapsed between the initial bleeding and the first clinical manifestations was evaluated. It varied from 3 to 30 days, with more than half of the patients bled during the first 2 weeks after onset. Controls for adults were normal blood donors. In two circumstances however-children and sequential studies (see below)-an age-matched healthy subject was bled with the patient.
/3,-MICROGLOBULIN
AND INFECTIOUS
MONONUCLEOSIS
57
3. Clq binding assay. It was performed as described by Zubler et al. (11). The Clq binding activity (Clq BA) was expressed as percentage of the TCAprecipitable counts of the 1251-labeled Clq introduced in the test tubes. RESULTS
1. Sp2m in I.M.
Patients
S fi2rn levels of normal children aged 5 to 10 were first compared to that of normal adults. No difference was found. All control values were therefore pooled and any test serum with a S j32m concentration higher than three standard deviations from the mean value (3 r SD = 1.32 + 0.36 mater) was considered as positive. Using this threshold (2.40 mg/liter) 24 of our 28 patients had an elevated S P2m level in their first (or unique) sample examined. As the patients were diagnosed or referred to us at different times after the clinical onset, the time elapsed between the initial symptoms and the first (or unique) bleeding was estimated. S p2m levels or individual sera were then plotted as a function of these estimations. No characteristic pattern of evolution over the 6-week period following the putative clinical onset could be drawn from these data (Fig. 1). As this could reflect the difficulty in accurately determining the actual starting point of the disease, S j32m values of the nine sequentially bled patients were also plotted as a function of time, with the first bleeding as origin. As shown on Fig. 2, all but one serum sample were initially positive and all but one had returned to normal 1 month later. 2. Relutionship
between S P2m and Clinical
Manifestations
Clinical subgroups were formed according to the presence of symptoms characteristic, although not specific, of I.M. None of the two following conditionsrash (3 times) or biological evidence of hepatitis (10 times)-resulted in a S P2m pattern differing from that observed in their absence. Patients with lymph node enlargement of such magnitude that a needle biopsy was performed prior to any Sp 2m (mg/l
) .
!
--
O-7
' B-14
t .
l
.
---------*--
'15-21 '22-28 '29-35 Days after onset*
'
FIG. 1. S P2m level, determined for each individual patient in the first (or unique) bleeding, as a function of time after clinical onset. ---- Represents the upper limit of normal as defined by the mean value of control sera + 3 SD. *, Clinically defined as the time at which the first symptom appeared.
58
LAMELIN SP2m
(mg/i
6-
1
ET
1
2 Order
AL.
of sequenhal
j
L
bleedings*
FIG. 2. S p2m level as a function of time in sequentially bled patients. *, Blood collected 2 weeks apart. ---- Represents the upper limit of normal (Fig. 1).
serological test, with the diagnosis of lymphoma in mind, may represent an exception, In three such cases, the S P2m level was at its highest (4.18, 5.29, and 6.58 mg/liter, respectively). 3. Relationship
of S @2m with Other Biological
Parameters
The determination of anti-EBV antibody titers, WBC differential counts, and Clq BA in the nine sequentially bled patients gave the following results. IgM anti-VCA antibodies, the hallmark of EBV primary infection, were found in all initial sera, and decreased thereafter (2/9 positive at Day 28). The mean percentage of large pyroninophilic cells, established on Ficoll-Isopaque separated leukocytes, decreased in parallel to S /32m mean levels, from the initial to the last bleeding (Table 1). When however, individual sera were considered, no correlation could be found between the two parameters. As this might be due to some bias introduced in the lymphocyte isolation procedure, patients were selected for whom S P2m and WBC differentials established on total blood were determined within less than 24 hr. In this subgroup of 13, no correlation could again be found between either the percentage, or the absolute number of large pyroninophilic cells on the one hand and the S /32m level on the other. Because of the absence of clear cut distinction between the two populations of large pyroninophilic cells and large lymphocytes, the possibility still existed that the S P2m level might reflect the pool of peripheral large mononuclear cell (percentage or absolute counts) in toto. No such correlation could be found in 29 samples from the sequentially studied patients.
/3,-MICROGLOBULIN
AND INFECTIOUS
59
MONONUCLEOSIS
TABLE 1 LARGE PYRONINOPHILIC CELLS, IgM ANTI-VCA ANTIBODY, S p2m, AND Clq BA IN SEQUENTIAL BLOOD SAMPLES FROM NINE I.M. PATIENTS
Percentage large pyroninophilic ceW (al SD) IgM anti-VCA Frequency of positive sera (titers 2 10) GMT’ (Extreme values) S /32m (mgiliter) (tl SD) Clq BA (%j) Frequency of positive sera Mean value of positive sera” (-+I SD)
Day 001
Day 14
Day 28
13.1 (25.69)
3.55 (53.59)
2.55 (k2.31)
919 12.6 (10-40) 3.60 (k1.14)
819 12.6 (<5-40) 2.85 (kO.60)
2/9 <5 (55-20) 1.99 (kO.33)
619 3.3 (k2.7)
419 0.64 (20.45)
l/9
” The day of the first bleeding was arbitrarily chosen as Day 0. h Counts established on coded, randomized, cytocentrifugea preparations of Ficoll-Isopaque separated leucocytes from I.M. patients and controls. ( Geometric mean titer. ‘I Calculations made on actual values after subtraction of the upper limit of normal (defined as mean value of six normal sera + 3 SD).
The C lq BA was determined in the samples collected from the nine sequentially bled patients. The test was positive in six of the initial sera (3.3 ? 2.7), four of the second (0.64 r 0.45), and one of the third. It was negative in the two patients bled a fourth time. The significance of circulating immune complexes during I.M. is not known. As they may result from B-cell polyclonal activation of which EBV is a potent inducer (12), it was worth examining what relationship existed in individual sera between Clq BA and S p2m. As shown in Table 2, 9 sera out of 27 gave dissociated results (5 positive for S P2m and 4 positive for Clq BA) suggesting that the two parameters are not strongly associated. TABLE 2 DISTRIBUTION OF S P2m AND Clq BA POSITIVE AND NEGATIVE SERA, IN THE FIRST (OR UNIQUE) BLEEDING OF 27 I.M. PATIENTS Clq BA”
-__ S p2mb +
-
+
3 5
4 15
CLValues 2 mean of six normal sera + 3 SD are positive. ’ Values 2 2.40 mg/liter are positive.
60
LAMELIN
ET AL.
DISCUSSION
In this study of 28 patients with biologically confirmed I.M. the hypothesis was made that, if T lymphocyte activation results in the release of S P2m, the S /32m level must be increased. Our results indicate that this is the case. Similar observation had already been mentioned in the screening of cancers and other diseases including lymphoproliferative disorders (13, Cooper, personal communication). In a situation where the T-cell compartment is modified both quantitatively (the percentage of E rosettes was 10 to 20% higher than that of age-sex matched controls: data not shown), and qualitatively (presence of large pyroninophilic cells), the S p2m level is, irrespective of age, above normal in most patients. The initial symptoms of I.M. are usually nonspecific and slowly progressive, making the determination of the clinical onset rather inaccurate. This difficulty very likely accounts for the lack of correlation between S P2m level and length of evolution at the time of first (or unique) bleeding. That this is no longer the case when sequential bleedings are available, indicates that the duration of the silent period between the biological and clinical onsets must be quite different from one patient to another. Neither the presence of a rash, nor the existence of biological liver dysfunction corresponded, in our experience, to any special trend in the S j32m level. An interesting observation however was made in three patients who underwent a needle biopsy for their adenopathy: their high S ,02m level is compatible with our working hypothesis. The increase in S P2m level definitely resulted in our patients from an augmented production but not from a decreased renal catabolism: there was neither proteinuria, nor any known evidence of renal insufficiency. An increased S P2m level is not, among viral diseases, specific of I.M. Thus it has been observed in viral hepatitis B and non-A non-B (14). This contrasts with A-virus and drug-induced hepatitis where it remains normal (S. Beorchia, personal communication). A likely interpretation of these data is that activation of the lymphoid system, but not liver cell lysis, is responsible for P2m release. Although leukocyte interferon has been shown to enhance S P2m levels in vivo (15) and to increase both the membrane-bound and -released P2m by Burkitt lymphoma cells in vitro (16), it is not known whether this mediator plays a role in the S P2m increase of viral hepatitis and I.M. S P2m was, of course, explored in lymphoproliferative disorders: an increase was reported in chronic lymphocytic leukemia, lymphoma (17), and myeloma. In this latter condition, the S /32m level was found to correlate with the disease staging (18) but not with the extent of infiltration of the bone marrow by the plasma cells (19). The finding of elevated S P2m levels in African patients with Burkitt’s lymphoma is somewhat more difficult to interpret (19). “Healthy” children from the same geographical area had S ,&2m levels higher than that of age-matched Caucasian children. This, and the rise in serum immunoglobulin level, are likely to result from various chronic infections. There is however no link between these two parameters (20). This observation, similar to that made in benign gammapathy
P,-MICROGLOBULIN
AND
INFECTIOUS
MONONUCLEOSIS
61
and in multiple myeloma, stresses again the independence of y-globulin synthesis and S /32m levels. The lack of correlation in individual sera, between S /32m and Clq BA, is in line with this conclusion. In the context of I.M., the increased S /32m level observed in our patients may well result from the T-cell activation characteristic of the acute phase. This interpretation fits with other data such as, in vitro, the high /32m release from lymphocytes stimulated by T mitogens (3), and, in vivo, the elevated /32m level of synovial fluid associated with T-cell infiltration in rheumatoid arthritis patients (21). This contrasts with the absence of P2m elevation in other inflammatory arthritis, such as that of gout (21). In the presence of a normal renal function, the determination of S P2m level would thus represent a useful nonspecific systemic marker of lymphocyte activation, especially in these common situations where most of the activated cells do reside in lymphoid organs. Chronic forms of I.M. (22) may well represent such a situation. ACKNOWLEDGMENTS We are grateful to Drs. R. Garrigue, J. Robert, C. Tissot, and J. J. Viala who referred their I.M. patients to us. and to Dr. 0. Gentil’homme for her reading of most of the blood smears. The technical assistance of Ms. A. Desbos, M. Flacher, and B. Jeune is acknowledged. This work has been supported in part by (GIS No. 122003) contract from the Centre National de la Recherche Scientifique and by a grant from the University of Lyon (Faculte Grange-Blanche).
REFERENCES 1. Rook, G. A., Carswell, J. W., and Stanford, J. L., C/in. Exp. Immunol. 26, 129, 1976. 2. Bernier, G. M., and Fanger, M. W., J. Irnmunol. 109, 407, 1973. 3. Kin. K., Kasahara, T., Itoh. Y.. Sakurabayaschi, I., Kaway, T., and Morita. M., Immunology 36, 47, 1979.
4. Henle, G.. Henle, W., and Diehl, V., Proc. Nat. Acad. Sci. USA 59, 94, 1968. 5. Purtilo, D. T., Lancer ii, 882, 1976. 6. Klein, G.. In “Oncogenesis and Herpes Viruses III” (G. de The and W. Henle. Eds.), p. 815, IARC Scientific publications No. 24, Lyon, 1978. 7. Sheldon, P. J., Papamichail, M., Hemsted, E. H.. and Holborow, E. J., Ln,~cet i, 1153, 1973. 8. Papamichail, M., Sheldon, P. J., and Holborow, E. J., Clin. Exp. Zmmunol. 18, 1, 1974. 9. Henle, W., Henle, G., and Horwitz, C. A., Human Patho/. 5, 551, 1974. 10. Vincent, C., and Revillard, J. P., J. Immunol. Methods 10, 253, 1976. 11. Zubler, R. H., Lange, G., Lambert, P. H., and Miescher, P. A., J. Immunol. 116, 232, 1976. 12. Bird. A. G., and Britton, S., Immunol. Rev. 45, 41, 1979. 13. Shuster, J.. Gold, P., and Poulik, M. D., C/in. Chim. Acta 67, 307, 1976. 14. Beorchia, S., Vincent, C., Revillard, J. P., and Trepo, C. Clin. Chim. Acra 109, 245, 1981. 15. Fridman, W. H., Lucero, M., Magdelenat, H., Billardon, C., Falcoff, E., and Pouillart, P., In “Symposium on New Trends in Human Immunology and Cancer Immunotherapy” (B. Serrou and C. Rosenfeld, Eds.), p. 865, Doin, Paris, 1980. 16. Fellous, M., Bono, R., Hyatil, F., and Gresser, I., Eur. J. Immund. 11, 524, 1981. 17. Amlot, P. L., and Adinolfi, M., Eur. J. Cancer 15, 791, 1979. 18. Cassuto, J. P.. Krebs, B. P.. Joyner, M. V., Viot, G., Dujardin, P., Auddy, P., and Massey&. R., Puthol. Bid. 26, 345, 1978. 19. Morel], A.. and Riesen, W., Acta Huemufol. 64, 87, 1980.
62
LAMELIN
ET
AL.
20. Cooper, E. H., and Kerruish, S., In “PZ-Microglobulin in Disease,” p. 19, Pharmacia Diagnostics, England, 1980. 21. Talal, N., Grey, H. M., Zwaifler, N.. Michalski. J. P.. and Daniels, T. E., Science 187, 1196, 1975. 22. Chang, R. S., In “Infectious Mononucleosis,” p. 102, G. K. Hall Medical Publishers, Boston, 1980. Received October 19, 1981; accepted with revisions January 12, 1982
IMMUNOLOGY
Elevation
AND
24, 55-62 (1982)
IMMUNOPATHOLOGY
of Serum &Microglobulin Infectious Mononucleosis
Levels
during
JEAN-PIERRELAMELIN,“,’ CLAUDE VINCENT,$ CLAUDE FONTAINE-LEGRAND,+ AND JEAN-PIERRE REVILLARDS Lahorntories of *Immunology und tvirology, Centre Lyon. and $Luhoratory of Immunology. ERA-CNRS
Nutional de la Recherche 782, H6pital E. Herriot.
Scienti$qw. Lyon, Franw
&Microglobulin (P2m), a potential marker of in vitro T-cell activation, was measured in the earliest available serum sample from 28 acute infectious mononucleosis (I.M.) patients, all free of any renal dysfunction. The serum fi2rn (S p2m) level was higher than that of matched-healthy controls in 21. No correlation was found with the time elapsed from clinical onset, the clinical symptoms, or the percentage and absolute number of large lymphocytes. The highest S fi2rn levels were observed in three I.M. patients with the largest adenopathies. In nine of the patients, three to four sequential bleedings were examined. The S /32m increased levels returned to normal within 30 days, along with the main biological parameters of I.M. (large pyroninophilic cells, IgM anti-VCA antibody, circulating immune complexes). However, no correlation could be established in individual sera among them. These findings, along with other reports, indicate that T-cell activation results in increased P2m release and elevated S P2m levels. Determination of S fl2rn may therefore provide a global estimate of T-cell activation in viva. not necessarily correlated with changes in lymphocyte subsets that are amenable to study in peripheral blood.
INTRODUCTION
The assessment of the stimulation of the immune system in viva by the detection of soluble substances released by lymphoid cells would be of great help in the evaluation of immune disorders in man. So far, this has been achieved only for the B-cell compartment, by measuring the level of serum immunoglobulins. Conversely, the degree of activation of the T compartment has mainly relied on the enumeration and functional in vitro evaluation of T cells collected from the peripheral blood. The limitation of this approach is illustrated by several situations in which a discrepancy between lymphoid tissue and peripheral blood lymphocytes has been documented (1). P,-Microglobulin (/32m), a 12,000 molecular weight protein, noncovalently linked to glycoproteins bearing class I histocompatibility antigens on almost all cell membranes but especially on lymphocytes, may be considered as a potential marker for evaluating T-cell activation. Bernier and Fanger (2) first demonstrated that /32m was mainly a product of T lymphocytes since it was produced by peripheral blood lymphocytes when stimulated by phytohemagglutinin but not by the pokeweed mitogen. More recently it I Address reprint requests to J.-P. Lamelin, Laboratoire Carrel, 69372 Lyon Cedex 2, France.
d’immunologie-CNRS,
Faculte Alexis
5.5
0090-1229/82/070055-08$01.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form resewed.
56
LAMELIN
ET
AL.
was shown that using mixed populations of T and B lymphocytes of known ratios, the amount of /32m released in the culture medium from activated T cells was much higher than that of their B counterpart. It was then concluded that P2m release reflected primarily T-cell activation (3). Infectious mononucleosis (I.M.) is a benign lymphoproliferative disorder resulting from the primary infection with the Epstein-Barr virus (EBV) (4). This disease is characterized by a proliferation and activation of a majority of T lymphocytes in response to the transformation by EBV of a small number of B lymphocytes (5,6). This disease was therefore considered as a good clinical model for assessing whether changes in serum P2m (S /32) levels may be observed in the context of this well-characterized T-cell activation (7, 8). MATERIALS
AND METHODS
1. Patients. Twenty-eight patients with biologically confirmed I.M. were entered in the present study. Twenty-one were males and seven were females. Due to a biased access to patients (referred to us from either a children or a military hospital) there were two age groups: the nine children ranged from 5 to 10 and the young adults from 17 to 24. Biological evidence for I.M. came from positive Monospot (3 cases) or Paul-Bunnell-Davidsohn reaction (22 cases). In 13 subjects, including the 3 patients not diagnosed by the above tests, anti-Epstein-Barr virus antibodies were titrated (9). The serological pattern characteristic of I.M. was present in all: presence of IgM antibodies against the EBV capsid antigen (VCA) in the absence of detectable response towards the EB nuclear antigen (EBNA). Overt clinical manifestations including adenopathy, splenomegaly, and liver involvement (biologically confirmed) were recorded retrospectively from the medical charts. None of the patients had any past history of renal disease. Blood urea was normal and proteinuria absent in all cases. 2. S /32m determination. S /32m was evaluated by a radioimmunoassay set up with our reagents (10). /32m purified from the urine of renal transplanted patients was iodinated (1251)by the chloramine-T method. High avidity anti-/32m serum was raised in rabbit. The complexes 1251P2m-anti-/32m formed in the presence or absence of the test serum were precipitated in liquid phase of a guinea pig antirabbit Ig serum. Actual values were obtained from a standard curve run in parallel. Nineteen patients were bled once. Thirteen of them had their white blood cells (WBC) counted (and the differential established) within less than 24 hr. Nine patients were bled sequentially, 2 weeks apart, three times, and, in two cases, four times. In this group of patients, the WBC differential count was determined on Ficoll-Isopaque separated cells. The time elapsed between the initial bleeding and the first clinical manifestations was evaluated. It varied from 3 to 30 days, with more than half of the patients bled during the first 2 weeks after onset. Controls for adults were normal blood donors. In two circumstances however-children and sequential studies (see below)-an age-matched healthy subject was bled with the patient.
/3,-MICROGLOBULIN
AND INFECTIOUS
MONONUCLEOSIS
57
3. Clq binding assay. It was performed as described by Zubler et al. (11). The Clq binding activity (Clq BA) was expressed as percentage of the TCAprecipitable counts of the 1251-labeled Clq introduced in the test tubes. RESULTS
1. Sp2m in I.M.
Patients
S fi2rn levels of normal children aged 5 to 10 were first compared to that of normal adults. No difference was found. All control values were therefore pooled and any test serum with a S j32m concentration higher than three standard deviations from the mean value (3 r SD = 1.32 + 0.36 mater) was considered as positive. Using this threshold (2.40 mg/liter) 24 of our 28 patients had an elevated S P2m level in their first (or unique) sample examined. As the patients were diagnosed or referred to us at different times after the clinical onset, the time elapsed between the initial symptoms and the first (or unique) bleeding was estimated. S p2m levels or individual sera were then plotted as a function of these estimations. No characteristic pattern of evolution over the 6-week period following the putative clinical onset could be drawn from these data (Fig. 1). As this could reflect the difficulty in accurately determining the actual starting point of the disease, S j32m values of the nine sequentially bled patients were also plotted as a function of time, with the first bleeding as origin. As shown on Fig. 2, all but one serum sample were initially positive and all but one had returned to normal 1 month later. 2. Relutionship
between S P2m and Clinical
Manifestations
Clinical subgroups were formed according to the presence of symptoms characteristic, although not specific, of I.M. None of the two following conditionsrash (3 times) or biological evidence of hepatitis (10 times)-resulted in a S P2m pattern differing from that observed in their absence. Patients with lymph node enlargement of such magnitude that a needle biopsy was performed prior to any Sp 2m (mg/l
) .
!
--
O-7
' B-14
t .
l
.
---------*--
'15-21 '22-28 '29-35 Days after onset*
'
FIG. 1. S P2m level, determined for each individual patient in the first (or unique) bleeding, as a function of time after clinical onset. ---- Represents the upper limit of normal as defined by the mean value of control sera + 3 SD. *, Clinically defined as the time at which the first symptom appeared.
58
LAMELIN SP2m
(mg/i
6-
1
ET
1
2 Order
AL.
of sequenhal
j
L
bleedings*
FIG. 2. S p2m level as a function of time in sequentially bled patients. *, Blood collected 2 weeks apart. ---- Represents the upper limit of normal (Fig. 1).
serological test, with the diagnosis of lymphoma in mind, may represent an exception, In three such cases, the S P2m level was at its highest (4.18, 5.29, and 6.58 mg/liter, respectively). 3. Relationship
of S @2m with Other Biological
Parameters
The determination of anti-EBV antibody titers, WBC differential counts, and Clq BA in the nine sequentially bled patients gave the following results. IgM anti-VCA antibodies, the hallmark of EBV primary infection, were found in all initial sera, and decreased thereafter (2/9 positive at Day 28). The mean percentage of large pyroninophilic cells, established on Ficoll-Isopaque separated leukocytes, decreased in parallel to S /32m mean levels, from the initial to the last bleeding (Table 1). When however, individual sera were considered, no correlation could be found between the two parameters. As this might be due to some bias introduced in the lymphocyte isolation procedure, patients were selected for whom S P2m and WBC differentials established on total blood were determined within less than 24 hr. In this subgroup of 13, no correlation could again be found between either the percentage, or the absolute number of large pyroninophilic cells on the one hand and the S /32m level on the other. Because of the absence of clear cut distinction between the two populations of large pyroninophilic cells and large lymphocytes, the possibility still existed that the S P2m level might reflect the pool of peripheral large mononuclear cell (percentage or absolute counts) in toto. No such correlation could be found in 29 samples from the sequentially studied patients.
/3,-MICROGLOBULIN
AND INFECTIOUS
59
MONONUCLEOSIS
TABLE 1 LARGE PYRONINOPHILIC CELLS, IgM ANTI-VCA ANTIBODY, S p2m, AND Clq BA IN SEQUENTIAL BLOOD SAMPLES FROM NINE I.M. PATIENTS
Percentage large pyroninophilic ceW (al SD) IgM anti-VCA Frequency of positive sera (titers 2 10) GMT’ (Extreme values) S /32m (mgiliter) (tl SD) Clq BA (%j) Frequency of positive sera Mean value of positive sera” (-+I SD)
Day 001
Day 14
Day 28
13.1 (25.69)
3.55 (53.59)
2.55 (k2.31)
919 12.6 (10-40) 3.60 (k1.14)
819 12.6 (<5-40) 2.85 (kO.60)
2/9 <5 (55-20) 1.99 (kO.33)
619 3.3 (k2.7)
419 0.64 (20.45)
l/9
” The day of the first bleeding was arbitrarily chosen as Day 0. h Counts established on coded, randomized, cytocentrifugea preparations of Ficoll-Isopaque separated leucocytes from I.M. patients and controls. ( Geometric mean titer. ‘I Calculations made on actual values after subtraction of the upper limit of normal (defined as mean value of six normal sera + 3 SD).
The C lq BA was determined in the samples collected from the nine sequentially bled patients. The test was positive in six of the initial sera (3.3 ? 2.7), four of the second (0.64 r 0.45), and one of the third. It was negative in the two patients bled a fourth time. The significance of circulating immune complexes during I.M. is not known. As they may result from B-cell polyclonal activation of which EBV is a potent inducer (12), it was worth examining what relationship existed in individual sera between Clq BA and S p2m. As shown in Table 2, 9 sera out of 27 gave dissociated results (5 positive for S P2m and 4 positive for Clq BA) suggesting that the two parameters are not strongly associated. TABLE 2 DISTRIBUTION OF S P2m AND Clq BA POSITIVE AND NEGATIVE SERA, IN THE FIRST (OR UNIQUE) BLEEDING OF 27 I.M. PATIENTS Clq BA”
-__ S p2mb +
-
+
3 5
4 15
CLValues 2 mean of six normal sera + 3 SD are positive. ’ Values 2 2.40 mg/liter are positive.
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DISCUSSION
In this study of 28 patients with biologically confirmed I.M. the hypothesis was made that, if T lymphocyte activation results in the release of S P2m, the S /32m level must be increased. Our results indicate that this is the case. Similar observation had already been mentioned in the screening of cancers and other diseases including lymphoproliferative disorders (13, Cooper, personal communication). In a situation where the T-cell compartment is modified both quantitatively (the percentage of E rosettes was 10 to 20% higher than that of age-sex matched controls: data not shown), and qualitatively (presence of large pyroninophilic cells), the S p2m level is, irrespective of age, above normal in most patients. The initial symptoms of I.M. are usually nonspecific and slowly progressive, making the determination of the clinical onset rather inaccurate. This difficulty very likely accounts for the lack of correlation between S P2m level and length of evolution at the time of first (or unique) bleeding. That this is no longer the case when sequential bleedings are available, indicates that the duration of the silent period between the biological and clinical onsets must be quite different from one patient to another. Neither the presence of a rash, nor the existence of biological liver dysfunction corresponded, in our experience, to any special trend in the S j32m level. An interesting observation however was made in three patients who underwent a needle biopsy for their adenopathy: their high S ,02m level is compatible with our working hypothesis. The increase in S P2m level definitely resulted in our patients from an augmented production but not from a decreased renal catabolism: there was neither proteinuria, nor any known evidence of renal insufficiency. An increased S P2m level is not, among viral diseases, specific of I.M. Thus it has been observed in viral hepatitis B and non-A non-B (14). This contrasts with A-virus and drug-induced hepatitis where it remains normal (S. Beorchia, personal communication). A likely interpretation of these data is that activation of the lymphoid system, but not liver cell lysis, is responsible for P2m release. Although leukocyte interferon has been shown to enhance S P2m levels in vivo (15) and to increase both the membrane-bound and -released P2m by Burkitt lymphoma cells in vitro (16), it is not known whether this mediator plays a role in the S P2m increase of viral hepatitis and I.M. S P2m was, of course, explored in lymphoproliferative disorders: an increase was reported in chronic lymphocytic leukemia, lymphoma (17), and myeloma. In this latter condition, the S /32m level was found to correlate with the disease staging (18) but not with the extent of infiltration of the bone marrow by the plasma cells (19). The finding of elevated S P2m levels in African patients with Burkitt’s lymphoma is somewhat more difficult to interpret (19). “Healthy” children from the same geographical area had S ,&2m levels higher than that of age-matched Caucasian children. This, and the rise in serum immunoglobulin level, are likely to result from various chronic infections. There is however no link between these two parameters (20). This observation, similar to that made in benign gammapathy
P,-MICROGLOBULIN
AND
INFECTIOUS
MONONUCLEOSIS
61
and in multiple myeloma, stresses again the independence of y-globulin synthesis and S /32m levels. The lack of correlation in individual sera, between S /32m and Clq BA, is in line with this conclusion. In the context of I.M., the increased S /32m level observed in our patients may well result from the T-cell activation characteristic of the acute phase. This interpretation fits with other data such as, in vitro, the high /32m release from lymphocytes stimulated by T mitogens (3), and, in vivo, the elevated /32m level of synovial fluid associated with T-cell infiltration in rheumatoid arthritis patients (21). This contrasts with the absence of P2m elevation in other inflammatory arthritis, such as that of gout (21). In the presence of a normal renal function, the determination of S P2m level would thus represent a useful nonspecific systemic marker of lymphocyte activation, especially in these common situations where most of the activated cells do reside in lymphoid organs. Chronic forms of I.M. (22) may well represent such a situation. ACKNOWLEDGMENTS We are grateful to Drs. R. Garrigue, J. Robert, C. Tissot, and J. J. Viala who referred their I.M. patients to us. and to Dr. 0. Gentil’homme for her reading of most of the blood smears. The technical assistance of Ms. A. Desbos, M. Flacher, and B. Jeune is acknowledged. This work has been supported in part by (GIS No. 122003) contract from the Centre National de la Recherche Scientifique and by a grant from the University of Lyon (Faculte Grange-Blanche).
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4. Henle, G.. Henle, W., and Diehl, V., Proc. Nat. Acad. Sci. USA 59, 94, 1968. 5. Purtilo, D. T., Lancer ii, 882, 1976. 6. Klein, G.. In “Oncogenesis and Herpes Viruses III” (G. de The and W. Henle. Eds.), p. 815, IARC Scientific publications No. 24, Lyon, 1978. 7. Sheldon, P. J., Papamichail, M., Hemsted, E. H.. and Holborow, E. J., Ln,~cet i, 1153, 1973. 8. Papamichail, M., Sheldon, P. J., and Holborow, E. J., Clin. Exp. Zmmunol. 18, 1, 1974. 9. Henle, W., Henle, G., and Horwitz, C. A., Human Patho/. 5, 551, 1974. 10. Vincent, C., and Revillard, J. P., J. Immunol. Methods 10, 253, 1976. 11. Zubler, R. H., Lange, G., Lambert, P. H., and Miescher, P. A., J. Immunol. 116, 232, 1976. 12. Bird. A. G., and Britton, S., Immunol. Rev. 45, 41, 1979. 13. Shuster, J.. Gold, P., and Poulik, M. D., C/in. Chim. Acta 67, 307, 1976. 14. Beorchia, S., Vincent, C., Revillard, J. P., and Trepo, C. Clin. Chim. Acra 109, 245, 1981. 15. Fridman, W. H., Lucero, M., Magdelenat, H., Billardon, C., Falcoff, E., and Pouillart, P., In “Symposium on New Trends in Human Immunology and Cancer Immunotherapy” (B. Serrou and C. Rosenfeld, Eds.), p. 865, Doin, Paris, 1980. 16. Fellous, M., Bono, R., Hyatil, F., and Gresser, I., Eur. J. Immund. 11, 524, 1981. 17. Amlot, P. L., and Adinolfi, M., Eur. J. Cancer 15, 791, 1979. 18. Cassuto, J. P.. Krebs, B. P.. Joyner, M. V., Viot, G., Dujardin, P., Auddy, P., and Massey&. R., Puthol. Bid. 26, 345, 1978. 19. Morel], A.. and Riesen, W., Acta Huemufol. 64, 87, 1980.
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20. Cooper, E. H., and Kerruish, S., In “PZ-Microglobulin in Disease,” p. 19, Pharmacia Diagnostics, England, 1980. 21. Talal, N., Grey, H. M., Zwaifler, N.. Michalski. J. P.. and Daniels, T. E., Science 187, 1196, 1975. 22. Chang, R. S., In “Infectious Mononucleosis,” p. 102, G. K. Hall Medical Publishers, Boston, 1980. Received October 19, 1981; accepted with revisions January 12, 1982