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Angioimmunoblastic lymphadenopathy with dysproteinemia
Angioimmunoblastic Dysproteinemia
Lymphadenopathy
with
A Paihogenetic Link Between Physiologic Lymphoid Proliferation and Malignant Lymphoma
AVRIJM IlARRY
Z.
HLUMING,
(:.
ANDREW
COHEN. SAXON.
M.D.’
Two patients with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) were studied. Both patients had marked increases in all three major immunoglobulin classes, and both lacked suppressor cell adtivity in vitro. These findings are consistent with the theory that AILD is a defectively regulated immune response to an unidentified antigen(s) and could provide clues to the pathogenesis of other lymphoproliferative disorders as well.
M.D.’ M.D.
I.os Angeles. Gllr/ornio
Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a benign disorder of lymphocyte proliferation [l-3]. It is characterized by the acute onset of generalized lymphadenopathy, hepatospleno,megaly, unexplained fever. sweats and weight loss. Histologically, there is obliteration of normal lymph node architecture including infiltration of the lymph node capsule, extranodal deposits in the splcerf. liver, bone marrow, dermis [4] and lungs [3,5.8]. The etiology of AILD is unknown, and the repobtcd median survival is 15 months [z]. The two patients with this disorder described herein were recently found to have absent suppressor ccl1 activity in vitro and profoundly increased serum concentrations of polyclonal immunoglobulins as well. They are presented in an attempt to provide possible clues to the pathogenesis of AILD and of related disorders of lymphocyte proliferation. PATIENTS Case 1. Ccntcr
AND METHODS
A 57 year old Caucasian on April
21.1977.
Iymphadcnopathy. cralizctl
miin was hospitalizccl
with an tight
week history
hep;ltosplenomcgaly.
crythematous
at St. joscph’s
of progressive
fever (tcmpcr;lturc!s
rash and pancytopcnia
(hemoglobin
Medical
gencralizetl
to lOl”F].
hcrnatocrit value 22 per cent. white blootl cell count 1,9Ot)/mm” with locyte,
1 mctamyclocytc.
lymphocytes. From the University of Southern School of Medicinc: and the: Division
California of Clinical
Immunology ;lnd Allergy. IJCLA blcdicihc. Los Angclcs. California.
School of This bapcr
was supported in part by NIH Grant Cn 12800. Koqwsts for rqwints should bc addressed to Dr. Avrum Aluming. * Prc.wht address:
16055 Ventura
535. Encino. California 91336. + I’rc:Jent adtl!oss: Medical Croup 5363 fI;llt)o;i
fornia 9131fi.
Blvd.. Suite of Encino.
11ouIcv;ml. Suite 445, Encino.
Cali-
specimen I,ukes
6 stall
2 monocytes
of
An axillilry
AIIII. Tht! sections
lymph
were rcvicwod
I myc-
leukocytes. 79
forms. 10 polymorphonuclear
and 1 cosinophil].
was chnractcristic
gcn-
level 7.6 g/100 ml,
notlo bps) by Dr. Rob-t
who concurred.
Serum globulin
protein
was 10.5 g/100
of 8.2 g/lo0
100 to 450 mg/llH)
ml. with
ml. lmmunoglobulin
ml], immunoglobulin
570 to 1.800 mg/lMl
an albumin
of 2.3 g/l00
ml and a
A was 1,450 mg/lCK) ml [normal G (I&)
ml] and immunoglobulin
3.400 mg/100 ml [normal M [IgM]
rang: rango
3,IHH) mg/lOl) ml [normal
range 60 to 280 mg/lOO ml]. Immunoclectrophorcsis the strum. tliscloscd mycloitl
Attempted
cliscloscd marrow
a hypcrcrtllular to crythrcritl
September
1979
[hl:E)
both kappa and Inmbdil
aspiration
marrow
with
yicldcd mildly
ratio of 5:l ilntl
light chains
a dry tap. Marrow
dccrcascd
markedly
in
hiops).
mcgnkaryocytcs.
a
irlc:rc:;lsc?cl rc:tic:ulin.
The American Journal of Medicine
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ANGIOBLASTIC LYMPHADENOPATHY WITH DYSPROTEINEMIA-BLUMING
Despite treatment with Cytoxan@, adriamycin, bleomycin and prednisone, the patient died with massive generalized lymphadenopathy and hepatosplenomegaly on June 29.1977. Postmortem examination disclosed massive plasma cell infiltration of the dura. pleura, lungs pericardium, heart. liver, spleen, gallbladder, duodenum, adrenal glands, kidneys, testes, retroperitoneal fat and bone marrow. Case 2. A 65 year old Caucasian woman was hospitalized at Encino Hospital on October 14,1977, with a four week history of generalized lymphadenopathy, hepatosplenomegaly, fever (temperature to 102OF) and a generalized pruritic, erythematous rash. The hemoglobin level was 10.7 g/100 ml, hematocrit value 33 per cent, white blood cell count 10,700/mm3, with a normal differential and a platelet count of 119,000/mm3. A cervical lymph node biopsy specimen was considered to be consistent with AILD. The slides were reviewed by Drs. Robert Lukes and Henry Rappaport who concurred with the diagnosis. Serum protein was 9.7 g/100 ml, with an albumin of 2.6 g/l00 ml and a globulin of 7.1 g/100 ml. IgA was 1,100 mg/lOO ml (normal range 170 to 410 mg/lOO ml), IgG 2,400 mg/lOO ml [normal range 900 to 1.500 mg/lOO ml], and IgM 3.700 mg/lOO ml (normal range 50 to 325 mg/lOO ml). Immunoelectrophoresis disclosed both kappa and lambda light chains in the serum. A bone marrow aspirate was mildly hypocellular with a M:E ratio of 25:l and 9 per cent plasma cells. Because of obvious disease progression, the patient received a four-day course of therapy with Alkeran”, 12 mg orally/day, and prednisone, 100 mg orally/day. Within five days of completing therapy the patient became afebrile, her lymphadenopathy and hepatosplenomegaly regressed, and her rash disappeared. Within six weeks her serum immunoglobulin levels returned to normal, and she received a second course of Alkeran and prednisone therapy. Her course was complicated by an aregenerative anemia, suggested by erythroid hypoplasia noted on her initial marrow aspirate and for which she was treated with oxymethalone, 150 mg/day for 11 months, with induction of a complete remission. She received a total of two courses of Alkeran and prednisone therapy, as already noted, and as of November 1978 was in a complete remission and receiving no medicine: her hemoglobin level was 16.4 g/100 ml, albumin was 4.2 g/l00 ml, and globulin 3.2 g/100 ml.
METHODS Lymphocyte Preparation and Separation Procedures. Peripheral blood lymphocyte suspensions were prepared by Ficoll@-Hypaque differential sedimentation of heparinized blood according to the method of Bijyum [7]. The blood was diluted with an equal volume of 0.15N saline solution, and the suspension was layered over 15 ml of Ficoll@-Hypaque (specific gravity 1,078 to 1,080). After centrifugation at room temperature (600 X G, 35 minutes), the interface cells were removed and washed three times in minimal essential medium. T and B lymphocyte fractions were separated by density sedimentation of spontaneous rosettes formed by T lymphocytes and sheep red blood cells (SRBC) that had been pretreated with 2-aminoethylisothiouronium [8]. Phagocytic cells were removed by incubation of cells (10 to 20 X 106/ml] with car-
422
September
1979
The American Journal of Medicine
ET AL.
bony1 iron solution (Lymphocyte Separator Reagent. Technicon Corporation. Tarrytown, N.Y.) for 30 minutes at 37% with vigorous agitation. The cells with ingested iron were then pelleted with a magnet. The supernatant containing the nonphagocytic cells was decanted and washed three times with minimal essential medium. T lymphocytes were enuIdentification of Cell Markers. merated by rosette formation between lymphocytes and untreated SRBC (E rosette) (91. Cells with a receptor for the Fc portion of IgG (Fc cells) were enumerated by rosette formation with trypsinized SRBC coated with hyperimmune rabbit anti-SRBC antibody (EA rosette) [lo]. Complement receptorbearing cells were enumerated by rosette formation with complement-coated zymosan particles [ll]. Cells with membrane-bound immunoglobulin were detected by direct immunofluorescence of fresh lymphocytes. Polyvalent rabbit antihuman immunoglobulin (Meloy Laboratories) was ultracentrifuged for 90 mintues at 65,000 X G on receipt and for 30 minutes at 10,000 X G just before use to remove any aggregated IgG that might have bound to Fc receptor cells. Before addition of the antiserums, the lymphocytes were incubated at 37% for 1 hour and then washed to remove any IgG which might have bound to Fc receptors in vivo [12]. Class specific (IgA, IgG, IgM) antiserums were shown to be monospecific by immunofluorescence against various fixed myeloma cells of known immunoglobulin class and monoclonal immunoglobulin myeloma proteins coupled to Sepharosem beads. Cells with cytoplasmic fluorescence were enumerated by direct immunofluorescence of fixed lymphocytes with the same reagents as those used for membrane fluorescence but at a fivefold greater dilution. Procedures employed for identification of these cell markers have been previously detailed 1131. Fractionated and recombined periphCulture Conditions. eral blood T and B lymphocytes were cultured in RPM1 1640 medium buffered with sodium bicarbonate and supplemented with 1-glutamine (10 mM), gentamycin (50 mg/ml] and 15 per cent heat-inactivated fetal calf serum (FCS). Final culture volume was 1.5 ml in 13 X 100 mm plastic tubes (Falcon no. 20271, containing pokeweed mitogen (PWM) (GIBCO, Grand Island, N.Y.) at a final dilution of l/100 v/v. The tubes were incubated in a humidified atmosphere at 37°C with 5 per cent carbon dioxide for five days. Sixteen hours before termination the cells were centrifuged into a pellet, the medium was collected, and the cells were resuspended in 0.5 ml of medium deficient in nonradioactive methionine and supplemented with 3%methionine 50 uCi/ml (specific activity 400 Ci/mM, New England Nuclear). For kinetic analysis of immunoglobulin production, culture supernatants were removed 8 hours after initiation of the culture and at subsequent 24-hour intervals. Radiolabeling was then performed as described. After the Measurement of Synthesized Immunoglobulin. final incubation, duplicate cultures were centrifuged (500 X G for 10 minutes), the culture medium was removed, and the lymphocytes were lysed. Nuclear and cellular debris were removed by high-speed centrifugation (20,000 X G for 30 minutes). Ten microliter aliquots of the resulting cytoplasmic supernatant were precipitated with trichloracetic acid (10 per cent) to obtain total radioactive protein estimates. Remaining culture medium and cytoplasmic supernatants were divided equally into tubes containing 5 ~1 of polyvalent antihuman
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ANGIOBLASTIC
LYMPHADENOPATHY
immunoglobulin antiserums and incubated for 20 minutes at room temperature. The resulting antigen-antibody complexes were removed by formalin-fixed Staphylococcus aureus bacteria (containing protein A, which binds to the Fc portion of IgG by the method of Kessler 1141).After four washes the radioactive proteins were eluted with 100 ~1 of 4 per cent SDS-GM urea. Twenty microliters of this radioactive protein solution were precipitated with trichloroacetic acid, and the radioactivity was determined by liquid scintillation counting. The remainder of the radioactive samples were either directly analyzed by 10 per cent SDS-polyacrylamide [SDS-PAGE] or reduced in dithiothreitol (0.05 m) and alkylated with iodoacetamide (0.1 M) before electrophoretic analysis. After electrophorcsis the radioactive immunoglobulin molecules were visualized by fluoro-autoradiography [15]. A marker sample containing radioiodinated (7251)mu (~1, gamma [r] and light chains was electrophoresed in parallel with each gel run for molecular weight determinations. Assay for T Lymphocyte Helper and Suppressor Activity. This method has been presented previously together with experiments demonstrating that B cells were capable of responding to allogeneic stimulation or suppression and that allogeneic T lymphocytes can provide this helper or suppressor activity [16]. The test entails culturing purified B lymphocytes
with an increasing number of T lymphocytes, both untreated and irradiated. and quantitating the immunoglobulin produced by each culture combination. RESULTS Case 1. Peripheral blood was drawn from the patient on lune 29,1%‘7. Surface marker assays identified 32 per cent of the circulating lymphocytes as T cells; 18 per cent were Fc receptor cells, 17 per cent were complement receptor cells, and only 3 per cent were membrane immunoglobulin-bearing cells (“true” B cells). However, 36 per cent of the circulating lymphocytes studied by immunofluorescence stained for cytoplasmic immunoglobulin; 21 per cent contained IgG, 10 per cent contained IgM, and 8 per cent contained IgA. The lymphoid cells in this patient’s peripheral blood were thus producing IgG, IgM and IgA. In spontaneous immunoglobulin biosynthetic experiments, his cells were shown to be spontaneously secreting large quantities of IgG, IgM and IgA over 24 hours. Normal circulating lymphocytes do not produce immunoglobulin under these conditions [16]. When the patient’s lymphocytes were assayed for suppressor activity against normal, allogeneic lymphocytes stimulated by pokeweed mitogen, no suppression of normal immunoglobulin production was noted. Addition of normal T cells in high titer inhibited normal B cell immunoglobulin production with a lo-fold excess of T cells resulting in a 28 per cent reduction. The patient’s T cells used in the same titer in the same assay system reduced normal B cell immunoglobulin production by only 7 per cent.
Case 2. Peripheral blood was drawn from the patient on December 6.1977. Surface marker assays identified 67 per cent of the circulating lymphocytes as T cells; 18
September
WITH
DYSPROTEINEMIA
---iXI IMING ET AL.
per cent were Fc receptor cells, 14 per cent carried complement receptors, and only 2 per cent were membrane immunoglobulin-bearing cells (“true” B cells), Her T dells markedly enhanced allogeneic B cell immunoglobulin production (two- to threefold increments over contro1 T lymphocyte helper activity). No evidence of suppression of immunoglobulin biosynthesis was observed, however, at high T to B cell ratios when normal suppression of B cell biosynthesis by allogeneic T cells was observed in control cultures (Figure 1). Blood was sent to Dr. Werner Henle for Epstein-Barr virus antibody titers. IgM titer was less than 1:lO; IgG was 1:160. Early antigen (EA) titer was less than 1:lO. EB nuclear antigen (EBNA) titer was 120. This pattern, indicative of past Epstein-Barr virus infection, is not unusual, suggesting that at least Epstein-Barr virus antigens were not implicated in the pathogenesis of this patient’s illness. COMMENTS
AILD is characterized by generalized lymphadenopathy in most or all patients [l,Z\, hepatosplenomegaly in approximately 50 per cent [l], and a maculopapular rash in approximately 50 per cent. Pruritis, present in both our patients, may or may not be evident. Symptoms usually but not always present include unexplained fever, sweats and weight loss. Of 15 patients described by Frizzera et al. [l], nine died within 18 months of onset of symptoms, and only five remained free of disease 15 to 46+ months from diagnosis. Of 32 patients described by Lukes and Tindle [2]. follow-up information is available on 28, of whom 18 died with a median survival of 15 months. Laboratory findings include hypergammaglobulinemia in 24 of 28 (86 per cent) patients studied [~,a]. In most cases the hypergammaglobulinemia has been polyclonal. One patient has been reported to have a monoclonal IgG spike [l], several have had polyclonal increases in only one or two of the three major immunoglobulin classes, and two had cryoglobulinemia [2,16] but without evidence of monoclonicity. Diagnosis rests on a characteristic histologic tetrad seen in biopsied nodes with includes: (I] pronounced
diffuse obliteration of the normal lymph node architecture [I]; (2) proliferation of arborizing small vessels [1,2]; (3) pleomorphic infiltrate rich in immunoblasts [1,2]; and (4) amorphous acidophilic interstitial material [1,2]. Multinucleated cells with the appearance of Reed-Sternberg cells have been reported by one group [I?‘] but were considered by others to be distinguishable from classical Reed-Sternberg cells [z]. The massive lymphadenopathy, markedly elevated serum levels of IgG, IgA and IgM, absence of suppressor cell activity, spontaneous polyclonal immunoglobulin biosynthesis in vitro and markedly disseminated infiltration of tissue by plasmacytoid cells all suggest that AILD is an often fatal unregulated immune reaction. Chronic lymphocytic leukemia, nodular forms of lymphoma and Hodgkin’s disease art: all classified as ma-
1979
The American Journal of Medicine
Volume 67
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ANGIOBLASTIC
LYMPHADENOPATHY
WITH DYSPROTEINEMIA-BLUMING
ET AL.
200
180
160
140
Normal B lymphocytor 120
dono.
Normal B Iymphocytoa + normal lIkg*n*lc
f lymphocytea.
too Normal B Iymphocytor f lymphocytor
+
from pattont, LB.
80
60
40
20
c
T Lymphocytes rddod par cultun
x lo6
Figure 1. Effect bf increasing numbers of normal or patient (Case 3) T lymphocytes on normal allogeneic B lymphocyte productiqn.
lignant lymphoproliferative disorders, yet all carry a better prognosis than AILD, which is reported to be a benign entity [1,2]. The three primary critera used to classify lymphoproliferative processes as benign or malignant include clinical course, histology and evidence of monoclonal proliferation. Clinical Course. In 1828 Craigie [la] described unexplained and progressive lymph node enlargement, providing the earliest known description of malignant lymphoma. “Either after repeated attacks of inflammation, alternating with resolution or with a slow and indistinct form of the disease. a gland or a cluster of glands gradually enlarges, and resisting all means of resolution becomes unusually hard . . The great hardness and the malignant tendency of this growth
424
September 197.9 The American Journal of Medicine
have procured for it from most authors the ominous names of scirrhus and cancer.” Clinical appearance of progressive course was thus the first criterion employed to categorize malignant lymphoma. It is, however, noteworthy that Craigie, a pathologist, went on to write 1‘ . . . though correct enough for all practical purposes, these epithets (i.e., scirrhus and cancer] are not justified by the anatomical characteristics.” Similarly, Brill et al. [19]. in their report, on what we now recognize as nodular lymphoma, noted that they initially believed they were dealing with a benign hyperplasia of the lymph nodes. Continued observation of the clinical course of their patients led them to revise this impression, however, and to conciude they were dealing with malignant neoplasms of relatively slow evolution.
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LYMPHADENOPATHY
The clinical course of AILD is suggestive enough of a malignant disorder that in the majority of reported cases the patients, including the two described herein, have been treated with potent antilymphoma modalities including radiotherapy, corticosteroids and alkylating agent chemotherapy despite histologic benignancy [l-3,17,20].
Histology.
Histologic criteria are currently the basis for a diagnosis of malignancy. In fact, AILD has been called benign because it does not satisfy histologic criteria for a diagnosis of malignant lymphoma. Frizzera et al. [l] labeled AILD benign on the basis of the following histologic findings: (1) “The peripheral sinuses and remnants of germinal centers could still be seen despite the pronounced obliteration of the nodal structure and involvement of the capsule.” Lukes and Tindle [z], however, reported “the process extended throughout the lymph node leaving only an occasional discernible sinusoid and in three cases (of 32) a few small residual follicles.” Using preservation of the peripheral or subcapsular sinus to differentiate benign from malignant conditions is questionable in light of Salvador, Harrison and Kyle’s [al] finding of subcapsular sinus obliteration in 11 of 11 lymph nodes taken from patients with heterophil antibody-documented infectious mononucleosis-all of whom recovered and survived disease-free from 2+ to 13+ years without specific therapy. (2) “The association of lymphocyte transformation with pronounced vascular proliferation does not accord with any known malignant lymphoma.” AILD is a newly described entity, not because of its clinical presentation (which is remarkably similar to Hodgkin’s disease) but because of its unique histologic picture. Given that this pidture is a previously undescribed one, its uniqueness should not be used as a criterion for either malignancy or benignancy. (3) “In extranodal sites, AILD is not infiltrative like leukemia nor does it have the nodular character of malignant lymphoma.” However, in the absence of a known infectious etiology--yet with reported deposits in the spleen, liver, dermis, kidneys, bone marrow and lung-the finding of extranodal deposits strongly suggests this disease is similar to other lymphomas. (4) “The end result of AILD is lymphocytic depletion with perivascular fibrosis and not tumoural replacement of organ structures.” This very pattern of diffuse fibrosis is characteristic of Hodgkin’s disease, which is classically assigned to the family of malignant lymphoma and treated accordingly. Although histologic criteria for a diagnosis of malignancy are the most reproducible criteria currently available, they should be treated as the useful tools that they are rather than as absolutes, and more reliable systems of classification, perhaps on the basis of pathogenesis, should be sought and developed. The concept that a maligMonoclonal Proliferation. nant tumor in man is the result of an aberrant or mutant
September
WITH
DYSPROTEINEMIA--BLCrMING
ET AL.
clonal proliferation of cells, cure of which necessitates excision or destruction of the entire clone, is a longstanding and widely accepted one. Fialkow et al. have used GGPD phenotype in heterozygote females to demonstrate the monoclonal nature of a variety of malignant tumors including chronic myelocytic leukemia [22], plasmacytoma [23] and Burkitt’s lymphoma [24]. In Hodgkin’s disease, however, the “tumor cell” has not yet been identified. A polymorphic cellular infiltrate is characteristic of this disease, and the Reed-Sternberg cell has been shown, in most cases, to be an end-stage cell incapable of replication [25]. Benign monoclonal gammopathy by its existence demonstrates that monoclonal proliferation is not synonymous with malignancy. At least one report has identified atherosclerosis as a disease of monoclonal proliferation [26]. Finally, although polyclonal gammopathy is unusual in neoplastic diseases other than hepatoma [27], it has been reported in seven patients with “plasmacytic sarcomatosis” [28], in eight cases of lymphoproliferative disorders [29,30], and in the macroglobulinemia-myeloma double gammopathy syndrome [31]. Since a diagnosis of malignancy is vital only insofar as it helps a physician choose appropriate therapy, confusion could be reduced if treatment were based more upon an understanding of lymphoma pathogenetic mechanisms than upon empiric observations and arbitrary classifications. The study of AILD may yield clues to the pathogenesis of a wide spectrum of lymphoproliferative disorders. Since the basic process of AILD appears, according to Lukes, to be a hyperimmune proliferation of the B cell system [2], an explanation for the exuberance of the B cell response might provide clues to rational and effective therapy. A model for the regulation of an immune response is illustrated in Figure 2. This model shows regulation of
Q “.
-::
I
T
cell
Figure 2.
1979
-b
I
Model for regulation of immune reactivity.
The American Journal of Medicine
Volume 67
425
ANGIOBLASTIC
LYMPHADENOPATHY
I
.--.
WITtl
UYSPROTEINE;LZIA--.BLIlMINC
ET AL
--
-BSA-
1
Cell mmun. BsA_
+
+
Lymph Incidence
0
0
DBA/L
BSA lmurah
66 %
+
OBA/2
I
Kruger, G. 1971
Figure 3. Experimental serum albumin.
pathogenesis
the immune response through feedback controlled by antibody. Mechanisms implicated in this regulatory process include blockade of lymphocyte antigen receptor by antigen-antibody complexes (3.21or by antibody to surface receptor immunoglobulins [33]. In addition, there apparently exists a subpopulation of T lymphocytes capable of suppressing an immune response (341. The role of suppressor cells in the regulation of the immune response has been recently reviewed by Waldman and Broder 1351. Suppressor cells have been shown to be associated with the decreased levels of normal immunoglobulin observed in patients with multiple myeloma 1351.Absence of such suppressor cells in a patient exposed to multiple antigen stimulation might lead to the marked polyclonal hypergammaglobulinemia characteristic of AILD. This lack of supprcssor cell activity was demonstrated in our patients with AILD and was, indeed, associated with marked polyclonal hypergammaglobulinemia. Baker [36] has demonstrated that the suppressor T cells that regulate antibody response, at least to thymic-independent antigens, limit the extent of B cell proliferation. AILD may be a hypcrimmunc response due to defective production or activity of suppressor ‘I’ cells. There are now experimental data supporting lack of suppressor T ccl1 activity as a pathogenetic mcchanism for the autoimmune phenomena observed in NZB mice (371. The positive Coombs’ test found in 67 per cent of tested patients with AILD [l] and the association with Guillain-Barre syndrome [38] and Hashimoto’s thyroiditis 1391are all suggestive of autoimmune phcnomenn with a similar pathogcncsis. A positive response to
426
September 1676
The American Journal of Medicine
of malignant
lymphoma.
BSA = bovine
therapy with Levamisolem [20,40], an agent shown capable of restoring defective T cell activity in certain situations [41], is also consistent with the concept that AILD is a syndrome arising from defective T cell regulation of the physiologic response to antigenic stimulation. Malignant lymphoma may be another point within the same spectrum as AILD. In 1971, Kruger [42] published a series of experiments supporting the concept that malignant lymphoma is the end product of unregulated immune response to a nononcogenic antigen. Kruger divided DBA/2 mice into three groups (Figure 3). The first group received bovine serum albumin daily by intraperitoncal injection for up to 18 months. The second group received immunosuppressive doses of Imuran@ daily in their drinking water, and the third group received both the bovine serum albumin injections and the oral Imuran. In the first group, circulating antibody and cellular immunity to bovine serum albumin developed. The second group did not receive any bovine serum albumin and, therefore, had no measurable immunologic response to this antigen, humoral or cellular. In the third group, normal circulating antibody ‘titers to bovine serum albumin developed but the mice had no cellular response to this antigen, probably because of Imuran suppression of T lymphocyte activity.’ Most interesting was Kruger’s observation that no lymphomas developed in either the antigenically stimulated mice in the first group or the immunosuppressed mice in the second group. In sharp contrast, the incidence of malignant lymphoma in the mice in third group, receiving both antigenic stimulation and immunosuppressive therapy,
Volume 67
ANGIOBLASTIC LYMPHADENOPATHY WITH DYSPROTEINEMIA-BLIJMING
was 66 per cent. It is not difficult to envision that an inhibitor of T lymphocyte activity, such as Imuran, when administered in the face of continued B cell stimulation by bovine serum albumin, could allow physiologic B cell proliferation to overwhelm the suppressed control arm by freeing the B cell response from T cell suppression. The end result would be malignant lymphoma, although there need be no intrinsic autonomy to the replicating lymphoid cells. The strong morphologic similarity of infectious mononucleosis and Hodgkin’s disease already noted [al], when considered in this context of defective T cell regulation of B cell reactivity, suggests that infectious mononucleosis is a lymphoma that regularly undergoes spontaneous regression [43]. Purtillo et al. [44] have described a geneticallytransmitted recessive lymphoproliferative syndrome, phetiotypic expressions of which include fatal infectious mononucleosis, immunoblastic sarcoma, American Burkitt’s lymphoma, acquired hypogammaglobulinemia and aplastic anemia. Autopsy findings in kindred males who died of infectious mononucleosis included depletion of thymocytes and reduction of the weight of the thymus gland by approximately 50 per cent, depletion of T cells in lymph nodes and spleen, plasmacytosis in hematopoietic organs, and infiltration of brain and viscera by lymphocytes and plasma cells. These findings, identical to those of a previously described kindred with fatal infectious mononucleosis [45], suggested to these investigators that fatal infectious mononucleosis may be the result of uncontrolled proliferation of infected B cells-that the primary event in the pathogenesis of these entities may
ET AL.
be defective regulation rather than autonomous proliferation. In this context a marked deficiency of T lymphocytes has already been reported in AILD [46], as has anergy [5,40,47], in addition to the characteristic B lymphocyte hyperactivity. Therapy should thus be directed in significant part to identifying the regulatory defect and correcting it, rather than attempting to destroy the replicating cells. A classification of lymphoproliferative disorders based on the possible regulatory defect implicated in their pathogenesis has been proposed [48]. Studies are underway to determine the mode or modes of action of suppressor cells [34,35] and of a soluble immune response suppressor [49]. In addition, a circulating inhibitor of immune regulation has been described and found to be an autologous antibody against suppressor T cells [5O]. The absence of suppressor cell activity in the two patients described in this report, coupled with the marked polyclonal hypergammaglobulinemia seen in both, suggests that a defect in suppressor cell regulation,of B lymphocyte proliferation is intricately implicated in the pathogenesis of their illness (AILD). Further studies of the mechanism of regulation will, it is hoped, replace histology with pathogcnesis as the touchstone for the diagnostic classification and treatment of the lymphoproliferative disorders. ACKNOWLEDGMENT We are deeply grateful to Martha Bluming torial assistance in preparing this paper.
for her edi-
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G. Moran EM, Rappaport H: Angio-immunoblastic fymphatlcnopathy with dysprotcincmia. Lancet 1: 1070, 1974. 2. Lukcs RI, ‘Tindle BH: Immunoblastic lymphadenopathy: a hyperimmune entity resembling Hodgkin’s disease. N Engl 1Med 292: 1, 1975. :3. l:rizzcra G. Moran EM, Rappaport H: Annie-immunoblastic lymphatlcnop;lthy. Diagnos/s and cli&tI course. Am J Mccl 59: 803. 1975. 4. Mcijcr CjLM. Scheffcr E. LZIUWGP. ct (11.:Skin biopsy in ;~ngiclimmrlnol,I;lslic lyml~h;rtlcnop;rthy. Lancct I: 771, 1978. 5. Iscman MD, Schwarz MI, St;lnfortl RE: Interstitial pncumania in angio-immunoblastic lymphadenopathy with clysprotcnemia. A case report with special histopathologic studies. Ann Intern Mcd 85: 752. IYX. ti. Wcisenburger D, Arniitagc J. Dick F: Immunoblastic Frizzcra
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7.
Ijoy&
A: Isolation of mononuclear
from human
blood. Scant1
9.
Saxon
I .I
l,
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syndrome. IG.
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A, Fcltihaus IL. Robbins RA:
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Zicghclboim j. Gale RP, Chin A, et al.: Antibody dependent cellular cytotoxicity mediated by non-T Iymphocytcs. Clin Immrmol Immunopathol 3: 193, 1974. Hubcr C, Wigzcll H: A simple rosette hssay for demonstration of comulemcnt rcceutor sitos usinn ctrmolement coat& zymosan
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hypocom-
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beads.
Errr i Immunol
5: 432, 1955.
RT, Fu SM. Hoffman T. ct al.: I& on lvmohocvtn surfaces. Technical problems and the sigGfican& ok a third cell population. 1Immunol .I 14: 1310. 1975. Saxcm A, Portis I: Lymphoitl sl~l)po~)l~l~ltictn changes in rcgional lymph nodes in squamous head and neck c;~ncc~r. Cancer Res 37: 1154, 1977. Kcsslcr SW: Rapid isolation of antlgcns from c;clls with il staphylococcal protein A-anlibotly iltlsorl)t>rrt:p;lrnmctc:rs of the interaction of ;rntit)trdy-ilntigcn c:omplcxcs with llrotcin A, J Immunol 115: 1617. 1975. Bonncr WM. Laskcv RA: A film dctcction mcthotl for Iritirlm-lahck?d prot&s and nucleic ;tcids in polyiicrylamide gels. Eur 1Biochem 46: 83. 1974. Saxon A, Stevens RII, Ashman RF: Kcgrll;~tion of immuno,qlobnlin production in human pcriphcri~l blood Icukocvtcs: _ &llril;lr interactions. j Immnnol 1’18:1872, 1977. Schultz DR, Yunis AA: Immunnbl,lstic I\-rn~)h;ltionot);\th~~ with mixed cryoglol,ulincmi;1. A tlt>tililt:tl c;~sc strltlq. N Engl 1Mcd 292: 8.1975. Craigic D: Elements of Gcncral and Patholr);:ic: Anatom!. Etlinbrireh. Adam Black. I>2511. 18X Winchester
Brill NE, I%ehr G, Rosenthlrl
N: Goncr,llizctl
giant
lymtlh
follicle hypcrplasia of lymph nodos and spleen. A hitherto undcscribed type. lAMA 84: f(i8. 1915.
September 1979 The American Journal of Medicine
Volume 67
427
ANGIOBLASTIC LYMPHADENOPATHY WITH DYSPROTEINEMIA-BLIJMING
20.
21.
22.
Ellegaard 1. Boesen AM: Restoration of defective cellular immunity by Lcvamisole in a patient with immunoblastic lymphadenopathy. Stand 1Haematoll7: 36, 1976. Salvador AH, Harrison EC )r, Kyle RA: Lymphadenopathy due to infectious mononucleosis. Its confusion with malignant lymphoma. Cancer 27: 1029,1971. Fialkow P). Gartlcr SM, Yoshida A: Clonal origin of chronic myelocytic leukemia in man. Proc Natl Acad Sci 58: 1468.
35.
36.
1967.
“< 7 24. “5. “6. “7. 28.
29.
30.
31.
428
Fialkow PJ, Martin CM. Klein G. et al.: Evicloncc for a clonal origin of head and neck tumors. Int 1 Cancer 9: 133. 1972. Finlkow P), Klein E, Klein G. et al.: lmmunoglobulin and G6PD as markers of cellular origin in Burkitt Ivmnhoma. ” . y ] Exp Med 138: 89,1973. Kaplan IIS: Hodgkin’s Disease, Harvard IJnivcrsity Press. Cambridge, Mass., 1972. p 44. Bt?nditt EP, Benditt )M: Evidence for a monoclonal origin of human atherosclerotic plaques. Proc Natl Acad Sci 70: 1753.1973. Fcno lio C. Pcrcncy A. Isohc T, ct al.: Hepatoma associated wit.a a marked ulasmncvtosis and uolvclonal hvoeraam.’ maglobulinemia. Am ] t&d 55: Iii. 1&‘3. Flandrin G. Daniel MT. El-Y& G. et al.: Sarcomatosis ranglionnaires dilfuscs a differentiation plasmacytaire &cc anemic hcmolytiquc autoimmune. Actual Hemat 6: 25. 1972. Kim A. Hellcr P. Rapoanort H: Monoclonal vammooathics associated with lymphoproliferative disorders. k morphologic study. Am ] Clin Pathol 59: 282, 1973. Guardia ). Pedreira )D, Vidal MT, et al.: Malignant lymphoma with olasmacvtoid differentiation and wlvclonal nammopaihy. Acta Haematol (Basel) 55: 346.‘19?6. Prudzanski W. Ilnderdown B, Silver EH. et al.: Macroglobulincmia-mycloma double gammopathy. A study of 4 casts and a review of the literature. Am ] Med 57: 259, 1974. Dicncr E. Feldman M: Relationship bctwccn antigen and antibody induced suppression of immunity. Transplant Rev 8: 76, 1972. Ramsicr 1I. Lindcmann ): Allotypic antihtrclics. Transplant Rev 10: 57. 1972. Bluming AZ. Lynch M). Kavanah M. et al.: Transformation
September
1979
The American Journal of Medicine
37. 38.
ET AL.
antigens on stimulated lymphocytes. 1Immunol114: 717. 1975. Waldman TA, Broder S: Supuressor cells in the relrulation of the immune response. Progress in Clinical Immunology. vol Ill. [Schwartz RS ed.1.I. New York. Grune & Stratton 1977. p i55. Baker P): Homcostatic control of antibody responses. A model based on the recognition of cell-associated antibody by regulatory T cells. Transplant Rev 26: 3, 1975. Kalus MJ: Immunoblastic lymphadenopathy. Arch Pathol Lab Med 100: 465.1976. Wntanahc H: Association of immunoblastic lymphadenopathy and Hashimoto’s thvroiditis. Ann Intern Med 87: 62. 1977.
39. 40. 41. 42.
Bankhurst AD, Williams RC: Cellular origins of autoantibody-a perplexing question. Am J Med-61: 303.1976. Bensa )C. Faure ), Martin H. et al.: Levamisole in angioimmunoblastic lymphadcnopathy. Lancet 1:1081,1976. Churchill WH It-. David )R: Lcvamisole and cell modiated immunity. N Engl] Mcd 289: 375,1973. Kruger G: Impeircd immunologic reactivity as a pacemaker for lymphoma dcvclopment. Verh Dtsch Ges Pathol55: 200. 1971.
43. 44. 45. 46. 47. 48. 49. 56.
Bluming AZ: Spontaneous regression ofsarcoma.Natl Cancer Inst Monogr 44: 55. 1976. Purtillo DT, DcFlorio D )r. Hutt LM. et al.: Phcnotypic cxpression of an X-linked lymphoprolifcrative syndrome. N Engl J Mcd 297: 1077.1977. Bar RS, De Lor CH. Clausen KP. et al.: Fatal infectious mononucleosis in a family. N Engl J Med 290: 363.1974. Palutke M. Khilanani P. W&e. R: Immunologic and electron-microscopic characteristics of a case of immunoblastic Iymphadenopathy. Am J Clin Pathol65: 929,1976. Kosmidis PA, Axelrod AR, Palacas C, et al.: Angioimmunoblastic Iymphadenopathy. A T-cell deficiency. Cancer 42: 447, 1978. Bluming AZ: Cancer: The eighth plague-a suggestion of pnthogcnesis. Isr I Med Sci 14: 192. 1978. Sagawa;‘A. Ahdou NI: Suppressor-cell dysfunction in systemic lupus crythcmatosus. 1Clin Invest 62: 789. 1978. Twjomcy 1). Laughter AIL Steinberg AD: A serum inhibition of immune regulation in patients with systemic lupus erythcmatous. 1Clin lnvcst 62: 713. 1978.
Volume 67
Lymphadenopathy
with
A Paihogenetic Link Between Physiologic Lymphoid Proliferation and Malignant Lymphoma
AVRIJM IlARRY
Z.
HLUMING,
(:.
ANDREW
COHEN. SAXON.
M.D.’
Two patients with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) were studied. Both patients had marked increases in all three major immunoglobulin classes, and both lacked suppressor cell adtivity in vitro. These findings are consistent with the theory that AILD is a defectively regulated immune response to an unidentified antigen(s) and could provide clues to the pathogenesis of other lymphoproliferative disorders as well.
M.D.’ M.D.
I.os Angeles. Gllr/ornio
Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a benign disorder of lymphocyte proliferation [l-3]. It is characterized by the acute onset of generalized lymphadenopathy, hepatospleno,megaly, unexplained fever. sweats and weight loss. Histologically, there is obliteration of normal lymph node architecture including infiltration of the lymph node capsule, extranodal deposits in the splcerf. liver, bone marrow, dermis [4] and lungs [3,5.8]. The etiology of AILD is unknown, and the repobtcd median survival is 15 months [z]. The two patients with this disorder described herein were recently found to have absent suppressor ccl1 activity in vitro and profoundly increased serum concentrations of polyclonal immunoglobulins as well. They are presented in an attempt to provide possible clues to the pathogenesis of AILD and of related disorders of lymphocyte proliferation. PATIENTS Case 1. Ccntcr
AND METHODS
A 57 year old Caucasian on April
21.1977.
Iymphadcnopathy. cralizctl
miin was hospitalizccl
with an tight
week history
hep;ltosplenomcgaly.
crythematous
at St. joscph’s
of progressive
fever (tcmpcr;lturc!s
rash and pancytopcnia
(hemoglobin
Medical
gencralizetl
to lOl”F].
hcrnatocrit value 22 per cent. white blootl cell count 1,9Ot)/mm” with locyte,
1 mctamyclocytc.
lymphocytes. From the University of Southern School of Medicinc: and the: Division
California of Clinical
Immunology ;lnd Allergy. IJCLA blcdicihc. Los Angclcs. California.
School of This bapcr
was supported in part by NIH Grant Cn 12800. Koqwsts for rqwints should bc addressed to Dr. Avrum Aluming. * Prc.wht address:
16055 Ventura
535. Encino. California 91336. + I’rc:Jent adtl!oss: Medical Croup 5363 fI;llt)o;i
fornia 9131fi.
Blvd.. Suite of Encino.
11ouIcv;ml. Suite 445, Encino.
Cali-
specimen I,ukes
6 stall
2 monocytes
of
An axillilry
AIIII. Tht! sections
lymph
were rcvicwod
I myc-
leukocytes. 79
forms. 10 polymorphonuclear
and 1 cosinophil].
was chnractcristic
gcn-
level 7.6 g/100 ml,
notlo bps) by Dr. Rob-t
who concurred.
Serum globulin
protein
was 10.5 g/100
of 8.2 g/lo0
100 to 450 mg/llH)
ml. with
ml. lmmunoglobulin
ml], immunoglobulin
570 to 1.800 mg/lMl
an albumin
of 2.3 g/l00
ml and a
A was 1,450 mg/lCK) ml [normal G (I&)
ml] and immunoglobulin
3.400 mg/100 ml [normal M [IgM]
rang: rango
3,IHH) mg/lOl) ml [normal
range 60 to 280 mg/lOO ml]. Immunoclectrophorcsis the strum. tliscloscd mycloitl
Attempted
cliscloscd marrow
a hypcrcrtllular to crythrcritl
September
1979
[hl:E)
both kappa and Inmbdil
aspiration
marrow
with
yicldcd mildly
ratio of 5:l ilntl
light chains
a dry tap. Marrow
dccrcascd
markedly
in
hiops).
mcgnkaryocytcs.
a
irlc:rc:;lsc?cl rc:tic:ulin.
The American Journal of Medicine
Volume 67
421
ANGIOBLASTIC LYMPHADENOPATHY WITH DYSPROTEINEMIA-BLUMING
Despite treatment with Cytoxan@, adriamycin, bleomycin and prednisone, the patient died with massive generalized lymphadenopathy and hepatosplenomegaly on June 29.1977. Postmortem examination disclosed massive plasma cell infiltration of the dura. pleura, lungs pericardium, heart. liver, spleen, gallbladder, duodenum, adrenal glands, kidneys, testes, retroperitoneal fat and bone marrow. Case 2. A 65 year old Caucasian woman was hospitalized at Encino Hospital on October 14,1977, with a four week history of generalized lymphadenopathy, hepatosplenomegaly, fever (temperature to 102OF) and a generalized pruritic, erythematous rash. The hemoglobin level was 10.7 g/100 ml, hematocrit value 33 per cent, white blood cell count 10,700/mm3, with a normal differential and a platelet count of 119,000/mm3. A cervical lymph node biopsy specimen was considered to be consistent with AILD. The slides were reviewed by Drs. Robert Lukes and Henry Rappaport who concurred with the diagnosis. Serum protein was 9.7 g/100 ml, with an albumin of 2.6 g/l00 ml and a globulin of 7.1 g/100 ml. IgA was 1,100 mg/lOO ml (normal range 170 to 410 mg/lOO ml), IgG 2,400 mg/lOO ml [normal range 900 to 1.500 mg/lOO ml], and IgM 3.700 mg/lOO ml (normal range 50 to 325 mg/lOO ml). Immunoelectrophoresis disclosed both kappa and lambda light chains in the serum. A bone marrow aspirate was mildly hypocellular with a M:E ratio of 25:l and 9 per cent plasma cells. Because of obvious disease progression, the patient received a four-day course of therapy with Alkeran”, 12 mg orally/day, and prednisone, 100 mg orally/day. Within five days of completing therapy the patient became afebrile, her lymphadenopathy and hepatosplenomegaly regressed, and her rash disappeared. Within six weeks her serum immunoglobulin levels returned to normal, and she received a second course of Alkeran and prednisone therapy. Her course was complicated by an aregenerative anemia, suggested by erythroid hypoplasia noted on her initial marrow aspirate and for which she was treated with oxymethalone, 150 mg/day for 11 months, with induction of a complete remission. She received a total of two courses of Alkeran and prednisone therapy, as already noted, and as of November 1978 was in a complete remission and receiving no medicine: her hemoglobin level was 16.4 g/100 ml, albumin was 4.2 g/l00 ml, and globulin 3.2 g/100 ml.
METHODS Lymphocyte Preparation and Separation Procedures. Peripheral blood lymphocyte suspensions were prepared by Ficoll@-Hypaque differential sedimentation of heparinized blood according to the method of Bijyum [7]. The blood was diluted with an equal volume of 0.15N saline solution, and the suspension was layered over 15 ml of Ficoll@-Hypaque (specific gravity 1,078 to 1,080). After centrifugation at room temperature (600 X G, 35 minutes), the interface cells were removed and washed three times in minimal essential medium. T and B lymphocyte fractions were separated by density sedimentation of spontaneous rosettes formed by T lymphocytes and sheep red blood cells (SRBC) that had been pretreated with 2-aminoethylisothiouronium [8]. Phagocytic cells were removed by incubation of cells (10 to 20 X 106/ml] with car-
422
September
1979
The American Journal of Medicine
ET AL.
bony1 iron solution (Lymphocyte Separator Reagent. Technicon Corporation. Tarrytown, N.Y.) for 30 minutes at 37% with vigorous agitation. The cells with ingested iron were then pelleted with a magnet. The supernatant containing the nonphagocytic cells was decanted and washed three times with minimal essential medium. T lymphocytes were enuIdentification of Cell Markers. merated by rosette formation between lymphocytes and untreated SRBC (E rosette) (91. Cells with a receptor for the Fc portion of IgG (Fc cells) were enumerated by rosette formation with trypsinized SRBC coated with hyperimmune rabbit anti-SRBC antibody (EA rosette) [lo]. Complement receptorbearing cells were enumerated by rosette formation with complement-coated zymosan particles [ll]. Cells with membrane-bound immunoglobulin were detected by direct immunofluorescence of fresh lymphocytes. Polyvalent rabbit antihuman immunoglobulin (Meloy Laboratories) was ultracentrifuged for 90 mintues at 65,000 X G on receipt and for 30 minutes at 10,000 X G just before use to remove any aggregated IgG that might have bound to Fc receptor cells. Before addition of the antiserums, the lymphocytes were incubated at 37% for 1 hour and then washed to remove any IgG which might have bound to Fc receptors in vivo [12]. Class specific (IgA, IgG, IgM) antiserums were shown to be monospecific by immunofluorescence against various fixed myeloma cells of known immunoglobulin class and monoclonal immunoglobulin myeloma proteins coupled to Sepharosem beads. Cells with cytoplasmic fluorescence were enumerated by direct immunofluorescence of fixed lymphocytes with the same reagents as those used for membrane fluorescence but at a fivefold greater dilution. Procedures employed for identification of these cell markers have been previously detailed 1131. Fractionated and recombined periphCulture Conditions. eral blood T and B lymphocytes were cultured in RPM1 1640 medium buffered with sodium bicarbonate and supplemented with 1-glutamine (10 mM), gentamycin (50 mg/ml] and 15 per cent heat-inactivated fetal calf serum (FCS). Final culture volume was 1.5 ml in 13 X 100 mm plastic tubes (Falcon no. 20271, containing pokeweed mitogen (PWM) (GIBCO, Grand Island, N.Y.) at a final dilution of l/100 v/v. The tubes were incubated in a humidified atmosphere at 37°C with 5 per cent carbon dioxide for five days. Sixteen hours before termination the cells were centrifuged into a pellet, the medium was collected, and the cells were resuspended in 0.5 ml of medium deficient in nonradioactive methionine and supplemented with 3%methionine 50 uCi/ml (specific activity 400 Ci/mM, New England Nuclear). For kinetic analysis of immunoglobulin production, culture supernatants were removed 8 hours after initiation of the culture and at subsequent 24-hour intervals. Radiolabeling was then performed as described. After the Measurement of Synthesized Immunoglobulin. final incubation, duplicate cultures were centrifuged (500 X G for 10 minutes), the culture medium was removed, and the lymphocytes were lysed. Nuclear and cellular debris were removed by high-speed centrifugation (20,000 X G for 30 minutes). Ten microliter aliquots of the resulting cytoplasmic supernatant were precipitated with trichloracetic acid (10 per cent) to obtain total radioactive protein estimates. Remaining culture medium and cytoplasmic supernatants were divided equally into tubes containing 5 ~1 of polyvalent antihuman
Volume 67
ANGIOBLASTIC
LYMPHADENOPATHY
immunoglobulin antiserums and incubated for 20 minutes at room temperature. The resulting antigen-antibody complexes were removed by formalin-fixed Staphylococcus aureus bacteria (containing protein A, which binds to the Fc portion of IgG by the method of Kessler 1141).After four washes the radioactive proteins were eluted with 100 ~1 of 4 per cent SDS-GM urea. Twenty microliters of this radioactive protein solution were precipitated with trichloroacetic acid, and the radioactivity was determined by liquid scintillation counting. The remainder of the radioactive samples were either directly analyzed by 10 per cent SDS-polyacrylamide [SDS-PAGE] or reduced in dithiothreitol (0.05 m) and alkylated with iodoacetamide (0.1 M) before electrophoretic analysis. After electrophorcsis the radioactive immunoglobulin molecules were visualized by fluoro-autoradiography [15]. A marker sample containing radioiodinated (7251)mu (~1, gamma [r] and light chains was electrophoresed in parallel with each gel run for molecular weight determinations. Assay for T Lymphocyte Helper and Suppressor Activity. This method has been presented previously together with experiments demonstrating that B cells were capable of responding to allogeneic stimulation or suppression and that allogeneic T lymphocytes can provide this helper or suppressor activity [16]. The test entails culturing purified B lymphocytes
with an increasing number of T lymphocytes, both untreated and irradiated. and quantitating the immunoglobulin produced by each culture combination. RESULTS Case 1. Peripheral blood was drawn from the patient on lune 29,1%‘7. Surface marker assays identified 32 per cent of the circulating lymphocytes as T cells; 18 per cent were Fc receptor cells, 17 per cent were complement receptor cells, and only 3 per cent were membrane immunoglobulin-bearing cells (“true” B cells). However, 36 per cent of the circulating lymphocytes studied by immunofluorescence stained for cytoplasmic immunoglobulin; 21 per cent contained IgG, 10 per cent contained IgM, and 8 per cent contained IgA. The lymphoid cells in this patient’s peripheral blood were thus producing IgG, IgM and IgA. In spontaneous immunoglobulin biosynthetic experiments, his cells were shown to be spontaneously secreting large quantities of IgG, IgM and IgA over 24 hours. Normal circulating lymphocytes do not produce immunoglobulin under these conditions [16]. When the patient’s lymphocytes were assayed for suppressor activity against normal, allogeneic lymphocytes stimulated by pokeweed mitogen, no suppression of normal immunoglobulin production was noted. Addition of normal T cells in high titer inhibited normal B cell immunoglobulin production with a lo-fold excess of T cells resulting in a 28 per cent reduction. The patient’s T cells used in the same titer in the same assay system reduced normal B cell immunoglobulin production by only 7 per cent.
Case 2. Peripheral blood was drawn from the patient on December 6.1977. Surface marker assays identified 67 per cent of the circulating lymphocytes as T cells; 18
September
WITH
DYSPROTEINEMIA
---iXI IMING ET AL.
per cent were Fc receptor cells, 14 per cent carried complement receptors, and only 2 per cent were membrane immunoglobulin-bearing cells (“true” B cells), Her T dells markedly enhanced allogeneic B cell immunoglobulin production (two- to threefold increments over contro1 T lymphocyte helper activity). No evidence of suppression of immunoglobulin biosynthesis was observed, however, at high T to B cell ratios when normal suppression of B cell biosynthesis by allogeneic T cells was observed in control cultures (Figure 1). Blood was sent to Dr. Werner Henle for Epstein-Barr virus antibody titers. IgM titer was less than 1:lO; IgG was 1:160. Early antigen (EA) titer was less than 1:lO. EB nuclear antigen (EBNA) titer was 120. This pattern, indicative of past Epstein-Barr virus infection, is not unusual, suggesting that at least Epstein-Barr virus antigens were not implicated in the pathogenesis of this patient’s illness. COMMENTS
AILD is characterized by generalized lymphadenopathy in most or all patients [l,Z\, hepatosplenomegaly in approximately 50 per cent [l], and a maculopapular rash in approximately 50 per cent. Pruritis, present in both our patients, may or may not be evident. Symptoms usually but not always present include unexplained fever, sweats and weight loss. Of 15 patients described by Frizzera et al. [l], nine died within 18 months of onset of symptoms, and only five remained free of disease 15 to 46+ months from diagnosis. Of 32 patients described by Lukes and Tindle [2]. follow-up information is available on 28, of whom 18 died with a median survival of 15 months. Laboratory findings include hypergammaglobulinemia in 24 of 28 (86 per cent) patients studied [~,a]. In most cases the hypergammaglobulinemia has been polyclonal. One patient has been reported to have a monoclonal IgG spike [l], several have had polyclonal increases in only one or two of the three major immunoglobulin classes, and two had cryoglobulinemia [2,16] but without evidence of monoclonicity. Diagnosis rests on a characteristic histologic tetrad seen in biopsied nodes with includes: (I] pronounced
diffuse obliteration of the normal lymph node architecture [I]; (2) proliferation of arborizing small vessels [1,2]; (3) pleomorphic infiltrate rich in immunoblasts [1,2]; and (4) amorphous acidophilic interstitial material [1,2]. Multinucleated cells with the appearance of Reed-Sternberg cells have been reported by one group [I?‘] but were considered by others to be distinguishable from classical Reed-Sternberg cells [z]. The massive lymphadenopathy, markedly elevated serum levels of IgG, IgA and IgM, absence of suppressor cell activity, spontaneous polyclonal immunoglobulin biosynthesis in vitro and markedly disseminated infiltration of tissue by plasmacytoid cells all suggest that AILD is an often fatal unregulated immune reaction. Chronic lymphocytic leukemia, nodular forms of lymphoma and Hodgkin’s disease art: all classified as ma-
1979
The American Journal of Medicine
Volume 67
423
ANGIOBLASTIC
LYMPHADENOPATHY
WITH DYSPROTEINEMIA-BLUMING
ET AL.
200
180
160
140
Normal B lymphocytor 120
dono.
Normal B Iymphocytoa + normal lIkg*n*lc
f lymphocytea.
too Normal B Iymphocytor f lymphocytor
+
from pattont, LB.
80
60
40
20
c
T Lymphocytes rddod par cultun
x lo6
Figure 1. Effect bf increasing numbers of normal or patient (Case 3) T lymphocytes on normal allogeneic B lymphocyte productiqn.
lignant lymphoproliferative disorders, yet all carry a better prognosis than AILD, which is reported to be a benign entity [1,2]. The three primary critera used to classify lymphoproliferative processes as benign or malignant include clinical course, histology and evidence of monoclonal proliferation. Clinical Course. In 1828 Craigie [la] described unexplained and progressive lymph node enlargement, providing the earliest known description of malignant lymphoma. “Either after repeated attacks of inflammation, alternating with resolution or with a slow and indistinct form of the disease. a gland or a cluster of glands gradually enlarges, and resisting all means of resolution becomes unusually hard . . The great hardness and the malignant tendency of this growth
424
September 197.9 The American Journal of Medicine
have procured for it from most authors the ominous names of scirrhus and cancer.” Clinical appearance of progressive course was thus the first criterion employed to categorize malignant lymphoma. It is, however, noteworthy that Craigie, a pathologist, went on to write 1‘ . . . though correct enough for all practical purposes, these epithets (i.e., scirrhus and cancer] are not justified by the anatomical characteristics.” Similarly, Brill et al. [19]. in their report, on what we now recognize as nodular lymphoma, noted that they initially believed they were dealing with a benign hyperplasia of the lymph nodes. Continued observation of the clinical course of their patients led them to revise this impression, however, and to conciude they were dealing with malignant neoplasms of relatively slow evolution.
Volume 67
ANGIOBLASTIC
LYMPHADENOPATHY
The clinical course of AILD is suggestive enough of a malignant disorder that in the majority of reported cases the patients, including the two described herein, have been treated with potent antilymphoma modalities including radiotherapy, corticosteroids and alkylating agent chemotherapy despite histologic benignancy [l-3,17,20].
Histology.
Histologic criteria are currently the basis for a diagnosis of malignancy. In fact, AILD has been called benign because it does not satisfy histologic criteria for a diagnosis of malignant lymphoma. Frizzera et al. [l] labeled AILD benign on the basis of the following histologic findings: (1) “The peripheral sinuses and remnants of germinal centers could still be seen despite the pronounced obliteration of the nodal structure and involvement of the capsule.” Lukes and Tindle [z], however, reported “the process extended throughout the lymph node leaving only an occasional discernible sinusoid and in three cases (of 32) a few small residual follicles.” Using preservation of the peripheral or subcapsular sinus to differentiate benign from malignant conditions is questionable in light of Salvador, Harrison and Kyle’s [al] finding of subcapsular sinus obliteration in 11 of 11 lymph nodes taken from patients with heterophil antibody-documented infectious mononucleosis-all of whom recovered and survived disease-free from 2+ to 13+ years without specific therapy. (2) “The association of lymphocyte transformation with pronounced vascular proliferation does not accord with any known malignant lymphoma.” AILD is a newly described entity, not because of its clinical presentation (which is remarkably similar to Hodgkin’s disease) but because of its unique histologic picture. Given that this pidture is a previously undescribed one, its uniqueness should not be used as a criterion for either malignancy or benignancy. (3) “In extranodal sites, AILD is not infiltrative like leukemia nor does it have the nodular character of malignant lymphoma.” However, in the absence of a known infectious etiology--yet with reported deposits in the spleen, liver, dermis, kidneys, bone marrow and lung-the finding of extranodal deposits strongly suggests this disease is similar to other lymphomas. (4) “The end result of AILD is lymphocytic depletion with perivascular fibrosis and not tumoural replacement of organ structures.” This very pattern of diffuse fibrosis is characteristic of Hodgkin’s disease, which is classically assigned to the family of malignant lymphoma and treated accordingly. Although histologic criteria for a diagnosis of malignancy are the most reproducible criteria currently available, they should be treated as the useful tools that they are rather than as absolutes, and more reliable systems of classification, perhaps on the basis of pathogenesis, should be sought and developed. The concept that a maligMonoclonal Proliferation. nant tumor in man is the result of an aberrant or mutant
September
WITH
DYSPROTEINEMIA--BLCrMING
ET AL.
clonal proliferation of cells, cure of which necessitates excision or destruction of the entire clone, is a longstanding and widely accepted one. Fialkow et al. have used GGPD phenotype in heterozygote females to demonstrate the monoclonal nature of a variety of malignant tumors including chronic myelocytic leukemia [22], plasmacytoma [23] and Burkitt’s lymphoma [24]. In Hodgkin’s disease, however, the “tumor cell” has not yet been identified. A polymorphic cellular infiltrate is characteristic of this disease, and the Reed-Sternberg cell has been shown, in most cases, to be an end-stage cell incapable of replication [25]. Benign monoclonal gammopathy by its existence demonstrates that monoclonal proliferation is not synonymous with malignancy. At least one report has identified atherosclerosis as a disease of monoclonal proliferation [26]. Finally, although polyclonal gammopathy is unusual in neoplastic diseases other than hepatoma [27], it has been reported in seven patients with “plasmacytic sarcomatosis” [28], in eight cases of lymphoproliferative disorders [29,30], and in the macroglobulinemia-myeloma double gammopathy syndrome [31]. Since a diagnosis of malignancy is vital only insofar as it helps a physician choose appropriate therapy, confusion could be reduced if treatment were based more upon an understanding of lymphoma pathogenetic mechanisms than upon empiric observations and arbitrary classifications. The study of AILD may yield clues to the pathogenesis of a wide spectrum of lymphoproliferative disorders. Since the basic process of AILD appears, according to Lukes, to be a hyperimmune proliferation of the B cell system [2], an explanation for the exuberance of the B cell response might provide clues to rational and effective therapy. A model for the regulation of an immune response is illustrated in Figure 2. This model shows regulation of
Q “.
-::
I
T
cell
Figure 2.
1979
-b
I
Model for regulation of immune reactivity.
The American Journal of Medicine
Volume 67
425
ANGIOBLASTIC
LYMPHADENOPATHY
I
.--.
WITtl
UYSPROTEINE;LZIA--.BLIlMINC
ET AL
--
-BSA-
1
Cell mmun. BsA_
+
+
Lymph Incidence
0
0
DBA/L
BSA lmurah
66 %
+
OBA/2
I
Kruger, G. 1971
Figure 3. Experimental serum albumin.
pathogenesis
the immune response through feedback controlled by antibody. Mechanisms implicated in this regulatory process include blockade of lymphocyte antigen receptor by antigen-antibody complexes (3.21or by antibody to surface receptor immunoglobulins [33]. In addition, there apparently exists a subpopulation of T lymphocytes capable of suppressing an immune response (341. The role of suppressor cells in the regulation of the immune response has been recently reviewed by Waldman and Broder 1351. Suppressor cells have been shown to be associated with the decreased levels of normal immunoglobulin observed in patients with multiple myeloma 1351.Absence of such suppressor cells in a patient exposed to multiple antigen stimulation might lead to the marked polyclonal hypergammaglobulinemia characteristic of AILD. This lack of supprcssor cell activity was demonstrated in our patients with AILD and was, indeed, associated with marked polyclonal hypergammaglobulinemia. Baker [36] has demonstrated that the suppressor T cells that regulate antibody response, at least to thymic-independent antigens, limit the extent of B cell proliferation. AILD may be a hypcrimmunc response due to defective production or activity of suppressor ‘I’ cells. There are now experimental data supporting lack of suppressor T ccl1 activity as a pathogenetic mcchanism for the autoimmune phenomena observed in NZB mice (371. The positive Coombs’ test found in 67 per cent of tested patients with AILD [l] and the association with Guillain-Barre syndrome [38] and Hashimoto’s thyroiditis 1391are all suggestive of autoimmune phcnomenn with a similar pathogcncsis. A positive response to
426
September 1676
The American Journal of Medicine
of malignant
lymphoma.
BSA = bovine
therapy with Levamisolem [20,40], an agent shown capable of restoring defective T cell activity in certain situations [41], is also consistent with the concept that AILD is a syndrome arising from defective T cell regulation of the physiologic response to antigenic stimulation. Malignant lymphoma may be another point within the same spectrum as AILD. In 1971, Kruger [42] published a series of experiments supporting the concept that malignant lymphoma is the end product of unregulated immune response to a nononcogenic antigen. Kruger divided DBA/2 mice into three groups (Figure 3). The first group received bovine serum albumin daily by intraperitoncal injection for up to 18 months. The second group received immunosuppressive doses of Imuran@ daily in their drinking water, and the third group received both the bovine serum albumin injections and the oral Imuran. In the first group, circulating antibody and cellular immunity to bovine serum albumin developed. The second group did not receive any bovine serum albumin and, therefore, had no measurable immunologic response to this antigen, humoral or cellular. In the third group, normal circulating antibody ‘titers to bovine serum albumin developed but the mice had no cellular response to this antigen, probably because of Imuran suppression of T lymphocyte activity.’ Most interesting was Kruger’s observation that no lymphomas developed in either the antigenically stimulated mice in the first group or the immunosuppressed mice in the second group. In sharp contrast, the incidence of malignant lymphoma in the mice in third group, receiving both antigenic stimulation and immunosuppressive therapy,
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ANGIOBLASTIC LYMPHADENOPATHY WITH DYSPROTEINEMIA-BLIJMING
was 66 per cent. It is not difficult to envision that an inhibitor of T lymphocyte activity, such as Imuran, when administered in the face of continued B cell stimulation by bovine serum albumin, could allow physiologic B cell proliferation to overwhelm the suppressed control arm by freeing the B cell response from T cell suppression. The end result would be malignant lymphoma, although there need be no intrinsic autonomy to the replicating lymphoid cells. The strong morphologic similarity of infectious mononucleosis and Hodgkin’s disease already noted [al], when considered in this context of defective T cell regulation of B cell reactivity, suggests that infectious mononucleosis is a lymphoma that regularly undergoes spontaneous regression [43]. Purtillo et al. [44] have described a geneticallytransmitted recessive lymphoproliferative syndrome, phetiotypic expressions of which include fatal infectious mononucleosis, immunoblastic sarcoma, American Burkitt’s lymphoma, acquired hypogammaglobulinemia and aplastic anemia. Autopsy findings in kindred males who died of infectious mononucleosis included depletion of thymocytes and reduction of the weight of the thymus gland by approximately 50 per cent, depletion of T cells in lymph nodes and spleen, plasmacytosis in hematopoietic organs, and infiltration of brain and viscera by lymphocytes and plasma cells. These findings, identical to those of a previously described kindred with fatal infectious mononucleosis [45], suggested to these investigators that fatal infectious mononucleosis may be the result of uncontrolled proliferation of infected B cells-that the primary event in the pathogenesis of these entities may
ET AL.
be defective regulation rather than autonomous proliferation. In this context a marked deficiency of T lymphocytes has already been reported in AILD [46], as has anergy [5,40,47], in addition to the characteristic B lymphocyte hyperactivity. Therapy should thus be directed in significant part to identifying the regulatory defect and correcting it, rather than attempting to destroy the replicating cells. A classification of lymphoproliferative disorders based on the possible regulatory defect implicated in their pathogenesis has been proposed [48]. Studies are underway to determine the mode or modes of action of suppressor cells [34,35] and of a soluble immune response suppressor [49]. In addition, a circulating inhibitor of immune regulation has been described and found to be an autologous antibody against suppressor T cells [5O]. The absence of suppressor cell activity in the two patients described in this report, coupled with the marked polyclonal hypergammaglobulinemia seen in both, suggests that a defect in suppressor cell regulation,of B lymphocyte proliferation is intricately implicated in the pathogenesis of their illness (AILD). Further studies of the mechanism of regulation will, it is hoped, replace histology with pathogcnesis as the touchstone for the diagnostic classification and treatment of the lymphoproliferative disorders. ACKNOWLEDGMENT We are deeply grateful to Martha Bluming torial assistance in preparing this paper.
for her edi-
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