Tohoku

J. exp.

Med.,

1977,

Ultrastructural

122,

129-141

Study

of

Immunoblastic

Lymphadenopathy NOBORU MATSUMOTO, TOKUHIRO ISHIHARA ,* HISAICHI FUJII, TADAHIKO SHIOMURA, KATSUYO YAMAUCHI , YOSHIMI Y AMASHITA,* FUMIYA UCHINO* and SHIRO MIWA

Department of Internal Medicine and *Department of Pathology, Y amnaguchi University School of Medicine, Ube 755

MATSUMOTO,N., ISHIHARA, T., Fuaii, H., SHIOMURA, T., YAMAUCHI, K ., YAMASHITA,Y., UCHINO, F. and MiwA, S. Ulltrastructural Study of Im.m.unoblastic Lymphadenopathy. Tohoku J. exp. Med., 1977, 122 (2), 129-141 An autopsy case of angio-immunoblastic lymphadenopathy with dysproteinemia (Frizzera et al. 1974) or immunoblastic lymphadenopathy (Lukes and Tindle 1975) is reported. Clinical pictures and morphologic characteristics of affected organs were typical of this disease. In spite of combination chemotherapy, the patient took a rapid fatal course. Post-mortem examinations disclosed involvement of the lymph nodes, liver, lungs, kidneys and skin. Cellular infiltrates in the kidney were more monomorphous, suggesting the potential for the development of immunoblastic sarcoma. Electron microscopies of the affected lymph nodes revealed the proliferation of immunoblasts characterized by moderate amount of clear cytoplasm with abundant polyribosomes and by large nuclei with prominent nucleoli. Undulated tubules associated with the endoplasmic reticulum and giant mitochondria with the centrally placed cristae were observed in occasional immunoblasts. Cytoplasmic fragments of immunoblasts and filamentous material among the cells were considered to correspond to the amorphous intercellular material seen in histologic sections. immunoblastic lym phadenopathy; electron microscopy; lymph nodes

Immunoblastic lymphadenopathy (Lukes and Tindle 1975) or angio immunoblastic lymphadenopathy with dysproteinemia (Frizzera et al. 1974) is a newly recognized disease entity characterized by generalized lymphadenopathy, hepatosplenomegaly, immunologic abnormalities such as polyclonal hyperglobuli nemia and/or Coomhs-positive hemolytic anemia, and acute constitutional symptoms. The histologic patterns of the involved lymph nodes and other tissues show a diagnostic triad; 1) diffuse infiltration by proliferating immuno blasts, plasmacytoid blasts, plasma cells and occasional histiocytes and eosinophils, with loss of normal lymph node architecture, 2) arborizing vascular proliferation, and 3) deposition of amorphous interstitial material (Frizzera et al. 1974; Lukes and Tindle 1975; Valdes and Blair 1976). Although the histologic characteristics of the affected organs have been well documented, the fine structures of the lymph nodes have appeared only in a recent report by Palutke and co-workers (1976). Received

for publication,

December

8, 1976. 129

130

N. Matsumoto

In this study, clinical

and

an autopsy

morphologic

ultrastructures

of the

et al.

case of immunoblastic

features

involved

is

lymphadenopathy

reported

lymph

with

a

special

with typical reference

to

the

nodes.

REPORT OF A CASE A 54-year-old throat, generalized

house wife was lymphadenopathy

first seen on March 17, 1975, with fever, cough, sore that initiated in the cervical region, macropapular

rash and facial edema of one month's duration. Before admission, she was treated local doctor without improvement. On admission, the patient was a slender woman appeared acutely ill. Physical examinations showed numerous enlarged lymph nodes

by a who with

tenderness in cervical, supraclavicular, submandibular, axillar and inguinal regions. Tonsils were markedly swollen bilaterally. The liver was felt 4 cm below the right costal margin. The spleen was not palpated. Macropapular rash without itching was noted on the chest wall, upper extremities, back and abdomen. Laboratory

data

hemoglobin

12.5

leukocyte

count

49.5%,

cell

showed

was

beta

mg/100

ml,

Direct

Coombs

IgA

and

reaction,

Weil-Felix

revealed

a mixed and The

had

capillaries,

or

and

and

and On

of

9,

1.

biopsy

Section

vascular

mixed

in

of

type

the

the

areas. reactive

mixed

with

cells

large

immunoblast

-

LE

cells

the

(Fig.

again

April

cells

3).

There Capsular

mixed stain

. •~

immunoblasts

time

, no of

cellular

Fig.

a

pyronine

4. and

Focal eosin. •~

multinucleated . •~

strong

mononuclear

focal

deposition by

of

these

mixed

definite diagnosis lyunphoreticular were

histologic

was tissue

suspected findings

infiltrate

and

marked

cells and a Reed

lymphocytes. -Sternberg

cell.

400. tend

to

accumulate

immunoblast

around with

the

capillary

pyroninophilic

ves

cytoplasm

Inset .

(X

Methyl

100.

deposition 400.

. were

100.

sels. shows

occasion a

large

disease and

of

ill-defined

were

invasion

, plasma resembling

superficially

the

per

Binucleated

exhibited

was

Hodgkin's

show

with 2).

cells

of

.

she

biopsy was proliferation

1 and

cells

proliferation

Schiff

biopsy

of

(Figs.

done

skin

beginning

4).

was

Paul-Bunnel

A

small

immature

that

ml.

around the was suspected

cytoplasm

At

2300

, rheumatoid ,

.

Reed-Sternberg

nodes

nodes acid

mg/100

, especially lymphoma

cells

cellularity lymph

2 .0

negative

plasmacytoid

cells

between

lymph

eosin. •~

large

(Fig.

the

Periodic infiltrate

Pyroninophilic

and and

axillary

cervical

,

plasmacytoid

occasional

or

right

binucleated and

cells

cells

among

noted

the

No

globulin IgG

, a cervical lymph-node normal architecture with

the

pyroninophilic

changes

of

2. Polymorphous I nset shows a

3.

also

material

proliferation.

Hematoxylin Fig.

was

was

lymphoma April

Fig.

Fig.

cells

intermediate

26

at

resembling

Plasma

methyl-green-pyronine,

amorphous

March

superficially

2).

dermis

malignant but

and

alpha-2 showed

test

were

marrow 6:1.

electrophoresis

IgD

antibodies,

the

while,

of

of

protein

and

test

in

plasma

nucleoli,

with

infiltrates

made

On loss by

cells

(Fig.

multinuclear

eosinophilic cellular

prominent large

encountered reaction

38•Ž.

showed

bone

hemagglutination

of

a

A ratio

4.0%,

ml

agglutination

for

infiltration

multinucleated

staining

to

nodes

mg/100

proliferation

afebrile

serum

globulin

anti-nuclear

involvement

3%.

Immunodiffusion

Toxoplasma

Brucella

and

remained

310 for

negative.

alpha-1

and

polymorphonuclears

erythroid and

406•~104/mm3,

31.3•~104/mm3

14%,

monocytes

31.2%).

Tests

and

appendages,

lymph

cytoplasm

ally

were

elevations

The

pale

negative.

forms

ml

was

count

myeloid:

g/100

48%,

IgM

lymphohistiocytic

temperature

7.5

globulin

ml,

reaction

skin

arborizing

mg/100

syphilis

patient

formed.

was

gamma

and a

count

platelet band

with

(albumin

290 was

29%

protein

and

cent, was:

marrow

6.6%

Erythrocyte

per

lymphocytes

Total

test

follows:

differential

gammopathy

arthritis

vessels

The

found.

globulin

as 36

normocellular

polyclonal

9.0%

were

hematocrit

13.5%,

disclosed

atypical

admission ml,

6,900/mm3.

eosinophils

aspiration

or

on

g/100

of eosinophilic

amorphous

material

among

the

cells

.

Hematoxylin

400) green

Immunoblastic

Lymphadenopathy

131

132

N. Matsumoto

et al.

Immunoblastic

Lymphadenopathy

133

essentially similar to those observed in the cervical lymph nodes . A lymphangiogram revealed retroperitoneal adenopathy suggestive of lymphoma . On April 19, the spleen was felt and both tonsils were markedly swollen causing a difficulty in swallowing . To confirm the diagnosis, exploratory laparotomy and splenectomy were performed on April 28. The spleen was 220 g in weight and stained sections showed a focal infiltrate in the peri arteriolar sheath areas (Fig. 5). The liver appeared normal but the biopsy specimen revealed the mixed cellular infiltration confined to the portal areas . Characteristic histologic findings of the lymph nodes, skin, liver and spleen were considered unusual for hitherto described lymphomas. Based on the unusual morphologic features of the affected organs , increasing levels of immunoglobulins (IgG 3500 mg/100 ml; IgA 360 mg/100 ml; IgM 400 mg/100 ml; IgD 6.0 mg/100 ml) and negative results of serological tests for infectious diseases or collagen diseases, a diagnosis of immunoblastic lymphadenopathy or angio immunoblastic lymphadenopathy with dysproteinemia was made , which was kindly confirmed by Prof. M. Kojima, Department of Pathology, Fukushima Medical College . She continued to have fever, rash and generalized lymphadenopathy . Administration of tetracyclin was not effective. Lymphadenopathy gradually increased in size and number , and at the beginning of June she had bull neck and edema of the face and upper extremities . A chest X-ray film disclosed bilateral pleural effusion, which was confirmed to be chylothorax. Chyloperitoneum developed later. Because of the severity of the illness , she was given Nitromin, 100 mg intravenously, vincristine, 2 mg intravenously, and prednisolone, 50 mg for three days. Chemotherapy seemed to be effective to reduce lymphadenopathy and her general condition was improved for a while , despite the persistent elevation of body temperature. At the end of June, lymphadenopathy became prominent again with pleural effusion and retention of chylous ascites. Her general condition rapidly deteriorated. Chemotherapy was instituted again with concomitant administration of antibiotics. All these treatments were ineffective and the patient expired on the 114th hospital day. Her terminal stage was characterized by advanced hypoalbum inemia (1.3 g/100 ml) and elevated levels of SGOT (142 units), LDH (2990 units) and serum alkaline phosphatase (479 units). Pertinent laboratory data during her hospital course are summarized in Table 1. Autopsy Findings:

The major findings at autopsy were as follows; chylothorax (800 ml on

TABLE 1.

Fig.

5.

Section

of

interstitial Schiff Fig.

6.

stain. •~ The

diffuse Fig.

7.

showing

in

the

retroperitoneal

Cellular

Typical

mixed

cellular

periarteriolar

lymph Hematoxylin

infiltrates

Inset

eosin. •~ 8.

spleen,

laboratory

data

infiltrate

lymphatic

and sheath

deposition

of

areas.

Periodic

amorphous acid

400.

necrosis.

ance.

Fig.

the

material

Representative

(•~

400)

in

the

shows

node

at

and

eosion. •~

kidney a

higher

autopsy

at

shows

autopsy,

magnification

marked

cellular

depletion

showing of

a

monomorphous

infiltrates.

appear

Hematoxylin

100. plasma

cells

with

well-developed

and

100.

endoplasmic

reticulum. •~

4240

and

-

134

N. Matsumoto

et al.

the left, and 1,000 ml on the right) and chyloperitoneum (700 ml). There was generalized lymphadenopathy, and section of the lymph nodes showed marked cellular depletion and diffuse necrosis which were mainly attributed to chemotherapy (Fig. 6). The right and left lungs weighed 380 g and 230 g, respectively. They were atelectatic and small whitish nodules were scattered on the pleura which showed infiltration by plasma cells, plasmacytoid cells and large mononuclear cells. The liver was 1,490 g in weight and a nutmeg appearance was noted on section. Centrilobular necrosis and focal involvement of portal areas by the mixed cellular infiltration were noted. The left kidney weighed 170 g and the right 200 g. Small whitish nodules were present in the cortex. These well-defined nodules consisted of plasmacytoid cells and immature mononuclear cells, and cellular infiltrates were more monomorphous than those seen in the lymph nodes (Fig. 7). The bone marrow was hypocellular and no infiltration of atypical plasmacytoid cells was noted. METERIAL AND METHODS For

electron

fixed

in

After

washing

and uranyl

cold

sectioned acetate

microscopy, 4.15% in

small

pieces

glutaraldehyde

the

same

with

an

and

lead

buffer

Ivan citrate,

of

biopsied

solution

in

solution,

Sorvall ‡Ub and

they

lymph 0.1

were

with

Thin a

(axillary

sodium

post-fixed

ultramicrotome. examined

nodes

M

Hitachi

in sections HS-8

region)

were

cacodylate,

pH

1%

tetroxide

osmium were

electron

stained

7.4.

with

microscope.

OBSERVATIONS

Electron microscopies of the affected lymph nodes revealed a wide variety of cellular proliferation. Typical plasma cells were usually seen in cluster and were characterized by their abundant rough endoplasmic reticulum (rER), as shown in Fig. 8. These plasma cells occasionally contained dense amorphous globules and crystalline structures in the distended rER. As seen in Figs. 9 and 10, large cells characterized by a loose chromatin pattern, one or two distinct nucleoli and a clear cytoplasm with many polyribosomes and strands of rER were estimated to correspond to immunoblasts described previously (Paltuke et al. 1976). These immunoblasts occasionally contained undulated tubules (Fig. 11) or giant mitochondria with the centrally placed cristae in their cytoplasm (Fig. 12). One of characteristic findings was the presence of numerous cytoplasmic fragments among the cells (Fig. 13). Morphologic features of these fragments were essentially similar to those of the cytoplasm of immunoblasts. In addition to the cytoplasmic fragments, intercellular deposition of filamentous material was noted in some areas (Fig. 14). It was presumed that intercellular amorphous material observed in histologic sections was composed of these filamentous material and the cytoplasmic fragments of immunoblasts at least in part. The precise nature and origin of the intercellular filamentous material were not determined . DISCUSSION

In recent years, an atypical lymphoproliferative disorder that closely resembles Hodgkin's disease has been described under the term "angioimmunoblastic lymphadenopathy with dysproteinemia" (Frizzera et al . 1974) or "immunoblastic lymphadenopathy" (Lukes and Tindle 1975). New additional cases have appeared

Immunoblastic

Fig.

9.

The

nucleoli the Fig.

10.

immunoblasts and

rieht Higher

polyribosomes

are

numerous

lower

characterized

polyribosomes.

field. •~

4,600

magnification

of

and

Lymphadenopathy

moderately

a immunoblast, developed

by

a

loose

Cytoplasmic

showing endoplasmic

135

chromatin fragments

clear

pattern,

prominent

(arrows)

cytoplasm

reticulum. •~

are

with 10,300

seen

numerous

at

136

Fig.

Fig.

N. Matsumoto

11.

A

with

the

12. 14,000

Giant

binucleated endoplasmic mitochondria

inununoblast

containing

reticulum. •~ with

et al.

undulated

tubules

(arrows)

associated

8,900 the

centrally

placed

cristae

in

an

immunoblast. •~

Immunoblastic

Fig.

13.

Cytoplasmic

immunoblast, Fig.

14. •~

Deposition 28,900

fragments, are

observed of

filamentous

Lymphadenopathy

which among

show the

material

the

cells. •~ is

same

137

characteristics

as

seen

in

the

9,900

occasionally

seen

in

the

intercellular

space.

138

N. Matsumoto

et al.

in recent literatures (Horne et al. 1974; Nomanbhoy and Prager 1974; Tangun et al. 1974; Abu-Zahra and McDonald 1975; Schultz and Yunis 1975; Moore et al. 1976; Palutke et al. 1976; Valdes and Blair 1976). This disease is usually seen in middle-aged or older individuals, and is characterized by generalized lymphadeno pathy, hepatosplenomegaly, skin rash, systemic symptoms and immunologic abnor malities such as polyclonal gammopathy, Coombs-positive hemolytic anemia or cryoglobulinemia. At present, the cause of the disease is unknown. Based on the histologic findings of the affected organs, Frizzera et al. (1974) have suggested that the disease is not a neoplastic process but represents a graft-versus-host reaction. Fatal cases of graft-versus-host disease in two infants after intrauterine and exchange trans fusion for Rh-incompatible hemolytic disease have been reported by Parkman et al. (1974). In their cases, clinical features and morphologic findings of the reticulo endothelial organs closely resemble those of immunoblastic lymphadenopathy. According to Lukes and Tindle (1975), the condition is a non-neoplastic hyperimmune proliferation of B-cell system that may be triggered by a hypersensitivity reaction to therapeutic agents. Recent immunohistologic study has suggested that the immunoblasts seen in this disease are of B-cell origin (Valdes and Blair 1976). As has been pointed out by Moore and co-workers (1976), the possibility of a viral cause cannot be excluded. It is noteworthy that rubella infection has preceeded the onset of immunoblastic lymphadenopathy by 3 months in a case reported by Palutke et al. (1976). Of another interest is the case described by Schlutz and Yunis (1975). They reported a case of this disease which had received liver extract for many years and serum antibody to the liver extract was demonstrated in this patient. They proposed that chronic antigenic stimulation with the liver extract had an important role in the development of the disease . In our case, no responsible agents or preceeding infections were evident except for injection of antiboitics which the patient received after the development of full clinical manifestation of the disease . Although the etiology of immunoblastic lymphadenopathy still remains unknown, the histologic features are not indicative of neoplastic process , because the affected lymph nodes or other organs show polymorphous cellularity and have distinct morphologic features which differ from those of hitherto described lym phomas. In Lukes and Tindle's series of 32 patients, 3 cases later manifested a monomorphous proliferation of immunoblasts and they prop osed the term "i mmunoblastic sarcoma" (Lukes and Tindle 1975) . They speculate that immunoblastic lymphadenopathy is a distinct clinical and morphologic entity which is situated in an intermediate position between benign compensatory immun oblastic reactions and lymphoma of imni uioblasts (immunobaastic sarcoma) . In our case, theekidney showed nodular infiltrates composed of more monomorphous cellularity, and a transitional form from immunoblastic lymphaden opathy to true neoplastic process was suggested. Absence of monoclonal gammopathy , however, was not i ndicative of immunoblastic sarcoma .

Immunoblastic

Lymphadenopathy

139

Overall ultrastructural characteristics of the affected lymph nodes reported herein are essentially similar to those described by Palutke et al. (1976). The immunoblasts are characterized by moderate amount of clear cytoplasm with numerous polyribosomes and varied amounts of rER. The chromatin pattern is loose and two or more prominent nucleoli are clearly visible. Various transitional forms of immunoblasts with varying amounts of rER and typical plasma cells are noted, suggesting a close functional and morphologic relationship between these two cells. The immunoblasts show cytoplasmic blebbing or small protrusions on the surface. As has been reported by Palutke et al. (1976), the cytoplasmic fragments derived from the immunoblasts are frequently present in the inter cellular space, and these fragments may partly correspond to the eosinophilic amorphous material seen in histologic sections. In addition, fine filamentous material is also observed among the cells, and these filaments may also represent the interstitial substance. Tubular inclusions have been shown in endothelial cells and lymphocytes in the affected lymph nodes (Palutke et al. 1976). In this study, the same tubular structure was not revealed, but undulated tubules associated with the endo plasmic reticulum were noted in immunoblasts (Fig. 11). Undulated tubules have been described in a variety of disorders, and their detailed morphology and cell types in which tubular arrays have been observed are summarized by Chandra (1968) and Uzman et al. (1971). Uzman and co-workers (1971) observed tubular arrays in tumor cells of Hodgkin's disease, cultured cells from lymph node or huffy coat from Hodgkin's disease, acute leukemia and infectious mononucleosis, and they suggested that these tubular inclusions might not be viral particles but might reflect cellular response to a broad range of stimuli. Chandra (1968) also takes the same view that undulated tubules are not viral in nature but represent morphologic manifestations of pathologic changes taking place within the affected cells. Clinical and morphologic resemblance of immunobalstic lymphadenopathy to Hodgkin's disease and appearance of undulated tubules in both conditions suggest that there may be some etiologic relationship between these two lymphoproliferative disorders. In spite of the distinct histologic pattern of the affected organs, the clinical course of this disease is quite varied. Some patients take a rapid fatal course and the others remain stable for a long time without any maintenance therapy. Thus, the disease seems to be heterogenous at least in the clinical course. Several therapeutic regimens were used in treating the patients. Moore et al. (1976) reported that no apparent difference in therapeutic regimens was seen between the patients who died and those who did not. According to Frizzera et al. (1974), cytotoxic agents and/or corticosteroids were useful in controlling the disease only in one-third of their cases. They suggest that the use of immunosuppressive drugs may be responsible for the severe infection observed in many patients at the terminal stage, and they recommend symptomatic treatment as a reasonable therapeutic approach in patients with this disease. Rappaport and Moran (1975)

140

N. Matsumoto

also

emphasize

are therapeutic

that

cytotoxic

agents

of choice

agents

agents

and corticosteroid

only for a short

term.

Autopsy

lymph

mainly

nodes

attributed

with to

complete therapy.

are

contraindicated

in immunoblastic

tion of cytotoxic the

et al.

were given

specimens

revealed

obliteration Massive

and

corticosteroids

lymphadenopathy. to our patient, a marked

of nodal

accumulation

cellular

architectures, of

Combina who responded

chylous

depletion which fluid

of were

in the

pleural and peritoneal cavities with advanced hypoalbuminemia seemed to be the result of generalized obstruction of nodal architectures. At present, management of patients lymphomas

with this disease is problematic, seems to be contraindicated,

desirable with careful severe infections.

follow-up

but usual chemotherapy for malignant and only supportive therapy may be

of the

clinical

course

and

protection

against

Acknowledgment

We are deeply indebted to Prof. M. Kojima, Department of Pathology, Fukushima Medical College, for his reviewing the lymph rode sections and for his advice for the diagnosis. Excellent technical assistance of Mr. M. Yamashita, Department of Pathology, Yamaguchi University School of Medicine, is also acknowledged. This work was supported by a Research of Health and Welfare, and by a Grant-in-Aid Education, Science and Culture, Japan.

Grant for Specific Diseases from the Ministry for Scientific Research from the Ministry of

References

1) 2) 3) 4) 5) 6) 7) 8)

9)

10) 11) 12) 13)

Abu-Zahra, H.T. & McDonald, D.B. (1975) Angioimmunoblastic lymphadenopathy with dysproteinemia. Lancet, 1, 114-115. Chandra, S. (1968) Undulating tubules associated with endoplasmic reticulum in pathologic tissues. Lab. Invest., 18, 422-428. Frizzera, G., Moran, E.M. & Rappaport, H. (1974) Angio-immunoblastic lymphadeno pathy with dysproteinemia. Lancet, 1, 1070-1073. Horne, C.H.W., Fraser, R.A. & Petrie, J.C. (1974) Angio-immunoblastic lymphadeno pathy with dysproteinemia. Lancet, 2, 291. Lukes, R.J. & Tindle, B.H. (1975) Immunoblastic lymphadenopathy. A hyperimmune entity resembling Hodgkin's disease. New, Eng. J. Med., 292, 1-8. Moore, S.B., Harrison, E.G. Jr. & Wailand, L.H. (1976) Angioimmunoblastic lymphadenopathy. Mayo Clin. Proc., 51, 273-280. Nomanbhoy, Y.T. & Prager, P.R. (1974) Angioimmunoblastic lymphadenopathy with dysproteinemia. Lancet, 2, 409. Palutke, M., Khilanani, P. & Weise, R. (1976) Immunologic and electronmicroscopic characteristics of a case of immunoblastic lymphadenopathy. Amer. J. clin. Path., 65, 929-941. Parkman, R., Mosier, D., Umansky, I., Cochran, W., Carpenter, C.B. & Rosen, F.S. (1974) Graft-versus-host disease after intrauterine and exchange transfusions for hemolytic disease of the newborn. New Eng. J. Med., 290, 359-363 . Rappaport, H. & Moran, E.M. (1975) Angio-immunoblastic (immunoblastic) lymphadenopathy. New Eng. J. Med., 292, 42-43. Schultz, D.R. & Yunis, A.A. (1975) Immunoblastic lymphadenopathy with mixed cryoglobulinemia. A detailed case study. New Eng . J. Med., 292, 8-12. Tangnn, Y., Saraebasi, Z., Peckelen, Y. & Inceman, S . (1974) Angio-immunoblastic lymphadenopathy with dysproteinemia. Lancet, 1 , 1345-1346. Uzman, B.G., Saito, H. & Kasac, M. (1971) Tubular arrays in the endoplastic

Immunoblastic reticulum

in human

tumor

cells.

Lymphadenopathy

Lab. Invest.,

141

24, 492-498.

14) Valdes, A.J. & Blair, O.M. (1976) Angioimmunoblastic lymphadenopathy with dysproteinemia. Immunologic and ultrastructural studies. Anger. J. clin. Path., 66, 551-559.