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Nerve growth factor receptor expression on dendritic reticulum cells in follicular lymphoid proliferations
Nerve Growth Factor Receptor Expression on Dendritic Reticulum Cells in Follicular Lymphoid Proliferations R. SCOTT STROBACH, MD, HIROKAZU NAKAMINE, MD, ANEAL S. MASIH, MD, JAMES LINDER, MD, AND DENNIS D. WEISENBURGER, MD
Using an antibody to the nerve growth factor receptor (NGFR), we examined dendritic reticulum cells (DRCs) immunohistochemically in 62 formalin-fixed, paraffin-embedded lymph nodes from patients with reactive follicular hyperplasia or with various types of lymphoma. A dendritic staining pattern within germinal centers was present in 25 of 26 routinely processed lymph nodeis with reactive follicular hyperplasia. In contrast, dendritic staining with anti-NGFR was present within neoplastic follicles in only three of 28 follicular lymphomas. Staining of benign, residual germinal centers with anti-NGFR was present in mantle zone lymphoma and Hodgkin’s disease. These findings suggest a palssible role for the NGFR in the maturation and/or activation of normal DRCs. The loss of NGFR expression in most follicular lymphomas indicates that DRCs are altered as part of the neoplastic process. The possibility that DRCs may play a role in the pathwgenesis of follicular lymphoma is suggested. HUM PATHOL 22:481-485. Copyright e 1991 by W.B. Saunders Com-
shown to recognize DRCS.~-’ Since the detection of these antigens,.with the exception of leukocyte common antigen, requires frozen tissue. the study of DRCs in formalin-fixed, paraffin-embedded tissue has not been possible. Recently. an antibody to the nerve growth factor receptor (NGFR) has been shown to mark DRCs in paraffin-embedded tissue ,n but the expression of this receptor on DRCs in normal and pathologic lymphoid processes has yet to be defined. The NGFR is a cell membrane and cytoplasmic protein with a molecular weight of 70 to 80 kd, and the gene for this molecule has recently been cloned and sequenced.“-” The NGFR is present on and required for the maturation of certain sympathetic and sensory neurons and other neural crest-derived cells. However, recent studies have also demonstrated the NGFR on cells in the central nervous system of primates, as well as in a variety of’ normal extraneural tissues and soft tissue neoplasms. l’L-‘.l Phenotypic alteration of DRCs in reactive and neoplastic follicular processes has been previously reported. .4cid cysteine-proteinase inhibitor (ACPI), which has been demonstrated in the DRCs of reactive lymphoid follicles, may be downregulated or lost in follicular lymphoma. li.16 Germinal center staining for IgM in reactive follicles, but not neoplastic follicles, has also been shown.*7 In our study, the expression of NGFR on the DRCs of reactive and neoplastic follicular lymphoid proliferations was evaluated. We also evaluated the usefulness of anti-N(;FR antibody in the differential diagnosis of follicular hyperplasia versus follicular lymphoma using paraffin-section immunohistochemistry. The possible implications of our findings with regard to the pathogenesis of follicular lymphoma are discussed.
pany
Dendritic reticulum cells (DRCs) are highly specialized cells which are derived from monocytei histiocyte precursors in the bone marrow, and which normally reside in the secondary follicles of lymph nodes and other peripheral lymphoid organs.‘-” The primary function of DRCs is thought to be the transportation and presentation of antigen to germinal center B cells.“.” Dendritic reticulum cells also serve as physica. support for the germinal center and surrounding mantle zone. Thus, DRCs play a major role in the structure and function of lymphoid follicles during the humoral immune response. Dendritic reticulum cells can be stained immunohistochemicallv with a number of monoclonal antibodies which recognize a variety of leukocyte antigens, including OKB7 (CD21). BA-2 (CDY). Leu-MS (CD14), HLADR (Ia), and leukocyte common antigen (CD45). In addition, the monoclonal antibody DRC- 1 was specifically devleloped for the immunohistochemical detection’of DRCs.-’ Recently, antibodies to certain basement membrane components, such as laminin, type IV collagen, and desmopakin 1 and 2, have also been
MATERIALS AND METHODS Paraffin-embedded tissue from 6’1 lymph nodes was obtained from the files of the Nebraska Lymphoma Registry and included 26 cases of reactive follicular hyperplasia, ?H cases of non-Hodgkin’s lymphomas-1 1 follicular large cell type (FLC), three follicular mixed cell type, six follicular small cleaved cell type (FSC), four small lymphocytic lymphomas (SL), and four mantle zone lymphomas (MZL)-as well as eight cases of Hodgkin’s disease (HD). All nonHodgkin’s lymphomas were classified according to a mod-
From the Department of Pathology and Microbiology. University of’ Nebraska Medical Center. Omaha. NE. .4ccepted for publication September -1. 1990. Key wwA: nerve growth factor receptor, dendritic- retie-ulum cell. follicular hyperplasia, follicular lymphoma. Address correspondence and reprint requests to R. Scott Strobath. MD, IDepartment of Pathology and Microbiology. Universit, of Nebraski-1 Medical Center, 600 S 42nd St. Omaha. NE 6819X. Copyright Q 1991 by W.B. Saunders Cornpan\ 00~6-~177~Y1/2205-0012f.‘,.00/0
481
HUMAN PATHOLOGY
Volume 22, No. 5 (May 1991)
ification of the Working Formulation. inTissues were fixed for 24 hours in 10% neutral buffered formalin and embedded in paraffin, since initial studies showed that the use of B54xed tissue resulted in considerable background staining. To demonstrate expression of the NGFR on DRCs in reactive and neoplastic processes, and to compare the distribution of the NGFR with that of the DRC-I antigen, we also performed a double immunoperoxidase study on frozen sections of human tonsil and eight of the above follicular lymphomas. Anti-DRC- 1 was obtained commercially (R4123, Dakopatts, Carpinteria, CA) and was applied at a dilution of 1:50. followed by biotinylated goat anti-mouse secondary antibody (1:400). and then by avidin-labeled peroxidase (ABC, Vector Laboratory Systems, Burlingame, CA). This reaction was developed with 3,3’ diaminobenzidine tetrahydrochloride with 3% hydrogen peroxide for 5 minutes to produce a brown product. Then a mouse monoclonal anti-NGFR (NCFR-5) was applied at a dilution of 1:50, again followed by biotinylated secondary antibody, and finally by an avidin-labeled alkaline plhophatase and substrate (Vector Laboratory Systems) to produce a red reaction product. Sections were incubated 1 hour at room temperature with both mouse monoclonal antibodies and 30 minutes with secondary antibodies and ABC complex: 5-minute rinses in phosphate-buffered saline were done between incubations. For the paraffin sections, an avidin-biotin-peroxidase complex method with minor modification was used.‘” Sections were cut at 3 to 4 km and baked on poly-L-lysine coated slides. Sections were digested with 0.4% pepsm in 0.01 normal hydrochloric acid for 20 minutes, and then incubated with horse serum (blocking antibody) for 5 minutes. Primary antibody (anti-NGFR) was then applied at a working dilution of 1:50 for 1 hour at room temperature. Sections were then incubated for 30 minutes at room temperature with biotinylated goat anti-mouse secondary antibody (1:400) and ABC complex (Vector Laboratory Systems) with 5-minute rinses in phosphate-buffered salme between incubations. A freshly prepared solution of 3,3’ diaminobenzidine tetrahydrochloride containing 3% hydrogen peroxide was applied for 5 minutes to develop the enzyme reaction. The immunohistochemical staining pattern of all follicles in each specimen was graded independently by two observers (R. S. S. and A. S. M.), and the results were averaged for each lymph node. Only dendritic staining, defined as a filamentous pattern between lymphoid cells within follicles, was graded. No dendritic staining was graded as 0; weak dendritic staining within lymphoid follicles was graded as 1 + ; focally intense dendritic staining or diffuse dendritic staining was graded as 2 + ; diffuse, intense dendritic staining was graded as 3+. In the lymphomas, the staining pattern of the neoplastic follicles or infiltrates was graded, whereas the staining of benign residual germinal centers was ignored.
Lymphoid cells were not stained. In a rare follicle exclusively red or brown dendritic staining was present. Seven of the eight follicular lymphomas for which frozen tissue was available showed dendritic staining only with anti-DRC-1 (brown). The dendritic staining was present within neoplastic follicles, and the staining intensity in the lymphomas ranged from 2 + to 3 + One lymphoma showed focal (I+ ) dendritic staining with anti-NGFR (red), as well as diffuse (3 +) dendritic staining with anti-DRC-1 (brown). The results of anti-NGFR staining of paraffin sections are summarized in Table 1. Positive staining was seen in 25 of 26 cases of follicular hyperplasia, of which nearly half showed intense, diffuse (3 + ) reactivity ( 12 cases). The staining was confined to the germinal centers (Fig 1, top right). Lymphoid cells in the germinal centers, mantle zones, and interfollicular areas did not stain with anti-NGFR. Significant variation in staining intensity, ranging from 1 + to 3 + within different follicles in the same lymph node, was noted in eight cases. In particular, follicles adjacent to the capsule frequently showed more intense dendritic staining than more centrally located follicles. Three cases of human immunodeficiency virus (HIV)associated follicular hyperplasia were included and showed from 1 + to 2 + dendritic staining with antiNGFR. Two of these had focal areas of follicular involution which showed diminished dendritic staining (Fig 1, bottom left). Thirty-six lymphomas, including eight cases of HD, were examined with anti-NGFR. A dendritic staining pattern in benign, residual germinal centers in the uneffaced portions of some lymph nodes was present. Cases of HD and MZL frequently had residual, benign germinal centers which reacted with antiNGFR. Reed-Sternberg cells, however, were uniThe neoplastic lymphoid cells in formly negative. MZL were negative as well, and typically surrounded the benign germinal centers. Three follicular nonHodgkin’s lymphomas (two FSC and one FLC) showed weak, focal (1 + ) reactivity within neoplastic follicles (Fig 1, bottom right). The remaining follicular lymphomas and the diffuse non-Hodgkin’s lymphomas showed no detectable dendritic staining. The pseudofollicular proliferation centers in the four small lymphocytic lymphomas, furthermore, were uniformly negative. DISCUSSION Our study was first prompted by the finding of dendritic staining with anti-NGFR of Peyer’s patches in an intestine involved by Crohn’s disease. Our initial intent was, therefore, to determine whether the antigen recognized by anti-NGFR-5 was the same or different from that recognized by anti-DRC-1, since the dendritic staining patterns were nearly identical. The results of our double immunoperoxidase method with anti-DRC and anti-NGFR on frozen tissue indicate the presence of the NGFR on DRCs in reactive
RESULTS The double immunoperoxidase method performed on frozen sections of hyperplastic human tonsil revealed 3 + staining for both anti-DRC-1 (brown) and anti-NGFR (red, Fig 1. top left). The majority of follicles showed a mixture of red and brown dendritic staining within germinal centers. 482
NGFR EXPRESSION ON DRCs (Strobach et al)
FIGURE 1. Nerve growth factor receptor expression on dendritic reticulum cells in paraffin-embedded follicular lymphoid proliferations. (Top left] Reactive lymph node showing a follicular dendritic pattern of both the nerve growth factor receptor (red] and the DRC-1 antigen (brown). (Irnmunoperoxidase stain; magnification x 125.) (Top right) Lymph node with florid reactive follicular hyperplasia showing predominantly cytoplasmic dendritic staining confined largely to the germinal center. (Immunoperoxidase stain; magnification x 125.) (Bottom left] Lymph Inode with human immunodeficiencyvirus-associated follicular involution showing weak dendritic expression of NGFR, suggesting its loss or down-regulation. (Immunoperoxidase stain; magnification x 125.) (Bottom right) Lymph node with follicular small cleaved cell lymphoma showing weak dendritic expression of NGFR: however, the majority of the follicular lymphomas were negative. (Immunoperoxidase stain; magnification x 125.)
t\lthough the meaning of this homology is unclear, the possibility that B cells and DR(:s are activated in a similar f&hi& or by a similar factor during the humoral immune response is a consideration. Alternatively, follicular involution, which occurs during the course of. HIV infection. is associated Cth the loss of follicle center B cells and DRCs.““~“:’ In our study, two cases of‘ HI V-associated follicular hyperplasia with follicular involution showed diminished reactivity with anti-NGFR, suggesting that DRCs are downregulated and/or lost during HIV infection of germinal centers. No dendritic reactivity with anti-NGFR was seen in 25 of 28 lymph nodes with follicular lymphoma. In two cases of’ FSC and in one case of FIL. weak reactivity was observed within some neoplastic follicles. These findings suggest that the NC;FRs on DRCs are down-regulated or lost during the neoplastic process. Similar results using anti-ACPI ant1 anti-IgM antibodies have been previously reported.]“-” The concurrent down-regulation or loss of several apparently unrelated proteins on or in DRC:s seems to indicate a
hyperpla:sia, in contrast to a lack of NGFR staining in follicular lymphoma. ‘These results suggest the presence of separate binding sites for anti-NGFR-5 and anti-DRC- 1, although the antigen recognizecl by antiDRC-1 is not known.’ ‘These results furthermore indicate that DRCs express the NGFR. which may be required for growth or activation of these cells. The intense expression of NGFR on DRCs in follicular hyperplasia suggests a possible role f’or the NCFR in the activation of DRCs or B cells. In the process of‘ follicular hyperplasia, DRCs bind and present antigen in the form of antigen-antibody complexes to the follicle center B cells, which then undergo clonal expansion.‘J The end result is the production of plasma cells and/or memory B cells that are specific for the stimulating antigen. .Interestingly. CDw40. a putative B-cell activation antigen, shows home sequence homology with the NVGFR.“‘,” Cross reactivity, of anti-N(GFR with this activation antigen did not hkelv occur in our study since no lymphoid cells in the reactive or neoplastic cases marked with ;tnti-NGFR in frozen or paraf~n-er7lbedded tissue. 483
HUMAN PATHOLOGY
TABLE I.
Volume 22, No. 5 (May 1991)
Nerve Growth Factor Receptor Expression on Dendritic Reticulum Cells in Routinely Processed Lymph Nodes
Histologic Classification Reactive Follicular hyperplasia Lymphoma Follicular small cleaved cell type Follicular mixed cell type Follicular large cell type Small lymphocytic lymphoma Mantle zone lymphoma Hodgkin’s disease
Grade* 0
If
1
7
4 3
2+
2 -
10
1
6
3+
I?
more information regarding the diagnostic usefulness of such staining. The intimate association of DRCs with the neoplastic B cells in follicular lymphoma is particularly intriguing. The possible role of DRCs in the pathogenesis of follicular lymphoma may provide a fruitful area for future investigation.
No. Positive
25126
-
-
216
-
-
o/3
-
-
l/l 1
4
-
-
-
o/4
4 8
_ _
_
_
o/4 O/8
Acknowledgment.
The
NGFR-5
was
courteously
vided by A. Ross, PhD, Worcester Foundation mental Biology, Shrewsbury, MA.
pro-
for Experi-
REFERENCES 1. Jaffe ES: Histiocytoses of lymph nodes: Biology and differdiagnosis. Semin Diagn Pathol 5:376-390. 1988 2. Szakal AR: Microanatomy of lymphoid tissue during humoral immune responses. Annu Rev Immunol 7:91-109, 1989 3. Weisenburger DD. Harrington DS, Armitage JO: B-cell neoplasia. A conceptual understanding based on the normal humoral immune response. Pathol Annu 25:99-l 15. 1990 4. Naiem M, Gerdes J, Abdulaziz Z. et al: Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue. J Clin Path01 36:167-175. 1983 5. Carbone A, Manconi R, Poletti A, et al: Heterogeneous immunostaining patterns of follicular dendritic reticulum cells in human lymphoid tissue with selected antibodies reactive with different cell lineages. HUM PATHOL 19:51-56, 1988 6. Carbone A, Poletti A, Manconi R, et al: Heterogeneous in situ immunophenotyping of follicular dendritic reticulum cells in malignant lymphomas of B-cell origin. Cancer 60:2919-2926. 1987 7. Gloghini A, Colombatti A, Bressan G. et al: Basement membrane components in lymphoid follicles: Immunohistochemical demonstration and relationship to the follicular dendritic cell network. HUM PATHOL 20:1001-1007. 1989 8. Thompson SJ, Schatteman CC. Gown AM, et al: A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue. Am J Chn Pathol 92:415-423, 1989 9. Marano N. Dietzschold B, Earley JJ, et al: Purification and amino terminal sequencing of human melanoma nerve growth factor receptor. J Neurochem 48:225-232. 1987 10. Ross AH, Crab P. Bothwell M. et al: Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies. Proc Nat1 Acad Sci USA 8 1:6681-6685,1984 II. Chao MV. Bothwell MA, Ross AH, et al: Gene transfer and molecular cloning of the human NCF receptor. Science 232:518-521. 1986 ential
* Grading of staining patterns: 0, no dendritic staining detectable; 1 + , weak or focal dendritic staining; 2 + , focal intense or diffuse weak dendritic staining; 3+. diffuse, intense dendritic staining. Results of various follicles in each lymph node were av-
eraged.
generalized down-regulation of DRCs in the follicular lymphomas. Ultrastructural evidence of functional attenuation of DRCs in follicular lymphoma has also been previously reported. 24 Thus, DRCs may play a role in the pathogenesis of follicular lymphomas by virtue of their inability to regulate or inhibit the proliferation of the neoplastic B cells. However, further studies are needed to confirm this hypothesis. Four cases each of SL and MZL were examined. In SL, no staining of pseudofollicular proliferation centers was observed, indicating either an absence of DRCs within pseudofollicular proliferation centers or a loss of NGFRs on DRCs which might be present. Our recent study of SL with anti-DRC- 1 suggests that there are no DRCs in pseudofollicular proliferation centers.25 In all four cases of MZL, reactivity of the benign residual germinal centers was observed. These results are consistent with previous conclusions that the neoplastic cells in MZL originate from normal mantle zone lymphocytes rather than germinal center cells.26 The use of anti-NGFR immunohistochemistry for the discrimination of follicular hyperplasia from follicular lymphoma should be approached with caution. The number of cases examined in this study is small, and staining appears to be significantly affected by the method and quality of fixation. However, the application of this marker in selected cases in which the distinction between benign and neoplastic germinal centers is unclear may be helpful, especially when intense dendritic reactivity is present, favoring a reactive process. In conclusion, additional studies of NGFR expression in reactive and neoplastic follicular proliferations are warranted and may further elucidate the role of DRCs in these processes, as well as provide
12. Schatteman CC, Gibbs L, Lanahan AA, et al: Expression of NCF receptor in the developing and adult primate central nervous system. J Neurosci 8:860-873, 1988 13. Garin Chesa P, Rettig WJ. Thomson TM, et al: Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues. .J Histochem Cytochem 36:3X3-389, 1988 14. Perosio PM, Brooks JJ: Expression of nerve growth factor receptor in paraffin-embedded soft tissue tumors. Am J Clin Path01 132:152-160, 1988 15. Alavaikko M, Aine R, Rinne A, et al: Behaviour of dendritic reticulum cells possessing immunoreactive acid cysteineproteinase inhibitor in human lymphoid secondary follicles and in follicular-centre cell lymphomas. Int J Cancer 35:319-325, 1985 16. Gerdes 1. Stein H. Mason DY, et al: Human dendritic reticulum cells of lymphoid follicles: Their antigenic profile and their identification as multinucleated giant cells. Virchows Arch .IS1_ 42: 161-172, 1983 LZ
17.Braylan KC. Rappaport nodular lymphomas as compared sias. Blood 42:579-589, 1973
484
H: Tissue immunoglobulins in with reactive folhcular hyperpla-
NGFR EXPRESSION ON DRCs (Strobach et al)
generalized 1986
1X. Nathwani RN: Classifying Non-Hodgkin’s lymphomas. in Berard C. Dorfman RF (eds): Malignant Lymphoma. International Academy 01’ Pathology Monograph No. 29. Baltimore, MD, U’illiams & Wilkins, 1987. pp 18-X0 19. Hsru SM. Raine L. Fanger H: Llse of avidin-biotin-peroxidase complex (ABC) in an immunoperoxidase technique: A comparison between ABC and unlabelled antibody (P.4P) procedure. J Histochem <:vtochem 29:577-X0. 1981 20. Braesch-Andersen S. Paulie S. Koho H. et al: Biochemical c-haracteristics and partial amino acid sequence of the receptor-like human B c-ell and c-arcinoma antigen CDw40. J Immunol 142:562567. 1989 2 I. Stamenkovic I. Clark EA. Seed B: A B-lymphocyte activaton molecule related to the nerve growth factor receptor and induced by cyrokines in carcinomas. EMBO J X:1403-1410, 1989 22. Tenner-Racz I(, Racz P. Bofill. M, et al: HTLV-III/LA\ viral antigens in lymph nodes of homosexual men with persistent
lymphadenopathy
and
AIDS.
Am J Pathol
123:9-15,
?3. Alavaikko Yf. Rinne A, Jarvinen M. et al: Damage to secondary lymphoid follicles in AIDS-related persistent generalized lymphadenopathy: as revealed by the behaviour of dendritic reticulum cells possessmg immunoreactive acid cvsteine-proteinase inhibitor. Virchows Arch [B] 51):299-31 1, 19% 24. Rademakers LHPM. Peters JPJ, \an Llnnik JAM: Histiocvtir and dendritic reticulum cells in follicular structures of follicular lymphoma and reactive hyperplasia. Virrhows Arch [B] 44:X598, lYH3 25. O’<:onnor SJ, Dale); DT. U’eisenburger DD: Pseudofollicular proliferation centers in small lymphocvtic Iymphoma lack dendritic reticulum cells. Mod Pathol 2:67.4. 1989 (ahstr) 26. Weisenburger DD: Mantle zone Ivmphoma. histologic study. Cancer 53: 1073-1080. 1983
485
An immuno-
Using an antibody to the nerve growth factor receptor (NGFR), we examined dendritic reticulum cells (DRCs) immunohistochemically in 62 formalin-fixed, paraffin-embedded lymph nodes from patients with reactive follicular hyperplasia or with various types of lymphoma. A dendritic staining pattern within germinal centers was present in 25 of 26 routinely processed lymph nodeis with reactive follicular hyperplasia. In contrast, dendritic staining with anti-NGFR was present within neoplastic follicles in only three of 28 follicular lymphomas. Staining of benign, residual germinal centers with anti-NGFR was present in mantle zone lymphoma and Hodgkin’s disease. These findings suggest a palssible role for the NGFR in the maturation and/or activation of normal DRCs. The loss of NGFR expression in most follicular lymphomas indicates that DRCs are altered as part of the neoplastic process. The possibility that DRCs may play a role in the pathwgenesis of follicular lymphoma is suggested. HUM PATHOL 22:481-485. Copyright e 1991 by W.B. Saunders Com-
shown to recognize DRCS.~-’ Since the detection of these antigens,.with the exception of leukocyte common antigen, requires frozen tissue. the study of DRCs in formalin-fixed, paraffin-embedded tissue has not been possible. Recently. an antibody to the nerve growth factor receptor (NGFR) has been shown to mark DRCs in paraffin-embedded tissue ,n but the expression of this receptor on DRCs in normal and pathologic lymphoid processes has yet to be defined. The NGFR is a cell membrane and cytoplasmic protein with a molecular weight of 70 to 80 kd, and the gene for this molecule has recently been cloned and sequenced.“-” The NGFR is present on and required for the maturation of certain sympathetic and sensory neurons and other neural crest-derived cells. However, recent studies have also demonstrated the NGFR on cells in the central nervous system of primates, as well as in a variety of’ normal extraneural tissues and soft tissue neoplasms. l’L-‘.l Phenotypic alteration of DRCs in reactive and neoplastic follicular processes has been previously reported. .4cid cysteine-proteinase inhibitor (ACPI), which has been demonstrated in the DRCs of reactive lymphoid follicles, may be downregulated or lost in follicular lymphoma. li.16 Germinal center staining for IgM in reactive follicles, but not neoplastic follicles, has also been shown.*7 In our study, the expression of NGFR on the DRCs of reactive and neoplastic follicular lymphoid proliferations was evaluated. We also evaluated the usefulness of anti-N(;FR antibody in the differential diagnosis of follicular hyperplasia versus follicular lymphoma using paraffin-section immunohistochemistry. The possible implications of our findings with regard to the pathogenesis of follicular lymphoma are discussed.
pany
Dendritic reticulum cells (DRCs) are highly specialized cells which are derived from monocytei histiocyte precursors in the bone marrow, and which normally reside in the secondary follicles of lymph nodes and other peripheral lymphoid organs.‘-” The primary function of DRCs is thought to be the transportation and presentation of antigen to germinal center B cells.“.” Dendritic reticulum cells also serve as physica. support for the germinal center and surrounding mantle zone. Thus, DRCs play a major role in the structure and function of lymphoid follicles during the humoral immune response. Dendritic reticulum cells can be stained immunohistochemicallv with a number of monoclonal antibodies which recognize a variety of leukocyte antigens, including OKB7 (CD21). BA-2 (CDY). Leu-MS (CD14), HLADR (Ia), and leukocyte common antigen (CD45). In addition, the monoclonal antibody DRC- 1 was specifically devleloped for the immunohistochemical detection’of DRCs.-’ Recently, antibodies to certain basement membrane components, such as laminin, type IV collagen, and desmopakin 1 and 2, have also been
MATERIALS AND METHODS Paraffin-embedded tissue from 6’1 lymph nodes was obtained from the files of the Nebraska Lymphoma Registry and included 26 cases of reactive follicular hyperplasia, ?H cases of non-Hodgkin’s lymphomas-1 1 follicular large cell type (FLC), three follicular mixed cell type, six follicular small cleaved cell type (FSC), four small lymphocytic lymphomas (SL), and four mantle zone lymphomas (MZL)-as well as eight cases of Hodgkin’s disease (HD). All nonHodgkin’s lymphomas were classified according to a mod-
From the Department of Pathology and Microbiology. University of’ Nebraska Medical Center. Omaha. NE. .4ccepted for publication September -1. 1990. Key wwA: nerve growth factor receptor, dendritic- retie-ulum cell. follicular hyperplasia, follicular lymphoma. Address correspondence and reprint requests to R. Scott Strobath. MD, IDepartment of Pathology and Microbiology. Universit, of Nebraski-1 Medical Center, 600 S 42nd St. Omaha. NE 6819X. Copyright Q 1991 by W.B. Saunders Cornpan\ 00~6-~177~Y1/2205-0012f.‘,.00/0
481
HUMAN PATHOLOGY
Volume 22, No. 5 (May 1991)
ification of the Working Formulation. inTissues were fixed for 24 hours in 10% neutral buffered formalin and embedded in paraffin, since initial studies showed that the use of B54xed tissue resulted in considerable background staining. To demonstrate expression of the NGFR on DRCs in reactive and neoplastic processes, and to compare the distribution of the NGFR with that of the DRC-I antigen, we also performed a double immunoperoxidase study on frozen sections of human tonsil and eight of the above follicular lymphomas. Anti-DRC- 1 was obtained commercially (R4123, Dakopatts, Carpinteria, CA) and was applied at a dilution of 1:50. followed by biotinylated goat anti-mouse secondary antibody (1:400). and then by avidin-labeled peroxidase (ABC, Vector Laboratory Systems, Burlingame, CA). This reaction was developed with 3,3’ diaminobenzidine tetrahydrochloride with 3% hydrogen peroxide for 5 minutes to produce a brown product. Then a mouse monoclonal anti-NGFR (NCFR-5) was applied at a dilution of 1:50, again followed by biotinylated secondary antibody, and finally by an avidin-labeled alkaline plhophatase and substrate (Vector Laboratory Systems) to produce a red reaction product. Sections were incubated 1 hour at room temperature with both mouse monoclonal antibodies and 30 minutes with secondary antibodies and ABC complex: 5-minute rinses in phosphate-buffered saline were done between incubations. For the paraffin sections, an avidin-biotin-peroxidase complex method with minor modification was used.‘” Sections were cut at 3 to 4 km and baked on poly-L-lysine coated slides. Sections were digested with 0.4% pepsm in 0.01 normal hydrochloric acid for 20 minutes, and then incubated with horse serum (blocking antibody) for 5 minutes. Primary antibody (anti-NGFR) was then applied at a working dilution of 1:50 for 1 hour at room temperature. Sections were then incubated for 30 minutes at room temperature with biotinylated goat anti-mouse secondary antibody (1:400) and ABC complex (Vector Laboratory Systems) with 5-minute rinses in phosphate-buffered salme between incubations. A freshly prepared solution of 3,3’ diaminobenzidine tetrahydrochloride containing 3% hydrogen peroxide was applied for 5 minutes to develop the enzyme reaction. The immunohistochemical staining pattern of all follicles in each specimen was graded independently by two observers (R. S. S. and A. S. M.), and the results were averaged for each lymph node. Only dendritic staining, defined as a filamentous pattern between lymphoid cells within follicles, was graded. No dendritic staining was graded as 0; weak dendritic staining within lymphoid follicles was graded as 1 + ; focally intense dendritic staining or diffuse dendritic staining was graded as 2 + ; diffuse, intense dendritic staining was graded as 3+. In the lymphomas, the staining pattern of the neoplastic follicles or infiltrates was graded, whereas the staining of benign residual germinal centers was ignored.
Lymphoid cells were not stained. In a rare follicle exclusively red or brown dendritic staining was present. Seven of the eight follicular lymphomas for which frozen tissue was available showed dendritic staining only with anti-DRC-1 (brown). The dendritic staining was present within neoplastic follicles, and the staining intensity in the lymphomas ranged from 2 + to 3 + One lymphoma showed focal (I+ ) dendritic staining with anti-NGFR (red), as well as diffuse (3 +) dendritic staining with anti-DRC-1 (brown). The results of anti-NGFR staining of paraffin sections are summarized in Table 1. Positive staining was seen in 25 of 26 cases of follicular hyperplasia, of which nearly half showed intense, diffuse (3 + ) reactivity ( 12 cases). The staining was confined to the germinal centers (Fig 1, top right). Lymphoid cells in the germinal centers, mantle zones, and interfollicular areas did not stain with anti-NGFR. Significant variation in staining intensity, ranging from 1 + to 3 + within different follicles in the same lymph node, was noted in eight cases. In particular, follicles adjacent to the capsule frequently showed more intense dendritic staining than more centrally located follicles. Three cases of human immunodeficiency virus (HIV)associated follicular hyperplasia were included and showed from 1 + to 2 + dendritic staining with antiNGFR. Two of these had focal areas of follicular involution which showed diminished dendritic staining (Fig 1, bottom left). Thirty-six lymphomas, including eight cases of HD, were examined with anti-NGFR. A dendritic staining pattern in benign, residual germinal centers in the uneffaced portions of some lymph nodes was present. Cases of HD and MZL frequently had residual, benign germinal centers which reacted with antiNGFR. Reed-Sternberg cells, however, were uniThe neoplastic lymphoid cells in formly negative. MZL were negative as well, and typically surrounded the benign germinal centers. Three follicular nonHodgkin’s lymphomas (two FSC and one FLC) showed weak, focal (1 + ) reactivity within neoplastic follicles (Fig 1, bottom right). The remaining follicular lymphomas and the diffuse non-Hodgkin’s lymphomas showed no detectable dendritic staining. The pseudofollicular proliferation centers in the four small lymphocytic lymphomas, furthermore, were uniformly negative. DISCUSSION Our study was first prompted by the finding of dendritic staining with anti-NGFR of Peyer’s patches in an intestine involved by Crohn’s disease. Our initial intent was, therefore, to determine whether the antigen recognized by anti-NGFR-5 was the same or different from that recognized by anti-DRC-1, since the dendritic staining patterns were nearly identical. The results of our double immunoperoxidase method with anti-DRC and anti-NGFR on frozen tissue indicate the presence of the NGFR on DRCs in reactive
RESULTS The double immunoperoxidase method performed on frozen sections of hyperplastic human tonsil revealed 3 + staining for both anti-DRC-1 (brown) and anti-NGFR (red, Fig 1. top left). The majority of follicles showed a mixture of red and brown dendritic staining within germinal centers. 482
NGFR EXPRESSION ON DRCs (Strobach et al)
FIGURE 1. Nerve growth factor receptor expression on dendritic reticulum cells in paraffin-embedded follicular lymphoid proliferations. (Top left] Reactive lymph node showing a follicular dendritic pattern of both the nerve growth factor receptor (red] and the DRC-1 antigen (brown). (Irnmunoperoxidase stain; magnification x 125.) (Top right) Lymph node with florid reactive follicular hyperplasia showing predominantly cytoplasmic dendritic staining confined largely to the germinal center. (Immunoperoxidase stain; magnification x 125.) (Bottom left] Lymph Inode with human immunodeficiencyvirus-associated follicular involution showing weak dendritic expression of NGFR, suggesting its loss or down-regulation. (Immunoperoxidase stain; magnification x 125.) (Bottom right) Lymph node with follicular small cleaved cell lymphoma showing weak dendritic expression of NGFR: however, the majority of the follicular lymphomas were negative. (Immunoperoxidase stain; magnification x 125.)
t\lthough the meaning of this homology is unclear, the possibility that B cells and DR(:s are activated in a similar f&hi& or by a similar factor during the humoral immune response is a consideration. Alternatively, follicular involution, which occurs during the course of. HIV infection. is associated Cth the loss of follicle center B cells and DRCs.““~“:’ In our study, two cases of‘ HI V-associated follicular hyperplasia with follicular involution showed diminished reactivity with anti-NGFR, suggesting that DRCs are downregulated and/or lost during HIV infection of germinal centers. No dendritic reactivity with anti-NGFR was seen in 25 of 28 lymph nodes with follicular lymphoma. In two cases of’ FSC and in one case of FIL. weak reactivity was observed within some neoplastic follicles. These findings suggest that the NC;FRs on DRCs are down-regulated or lost during the neoplastic process. Similar results using anti-ACPI ant1 anti-IgM antibodies have been previously reported.]“-” The concurrent down-regulation or loss of several apparently unrelated proteins on or in DRC:s seems to indicate a
hyperpla:sia, in contrast to a lack of NGFR staining in follicular lymphoma. ‘These results suggest the presence of separate binding sites for anti-NGFR-5 and anti-DRC- 1, although the antigen recognizecl by antiDRC-1 is not known.’ ‘These results furthermore indicate that DRCs express the NGFR. which may be required for growth or activation of these cells. The intense expression of NGFR on DRCs in follicular hyperplasia suggests a possible role f’or the NCFR in the activation of DRCs or B cells. In the process of‘ follicular hyperplasia, DRCs bind and present antigen in the form of antigen-antibody complexes to the follicle center B cells, which then undergo clonal expansion.‘J The end result is the production of plasma cells and/or memory B cells that are specific for the stimulating antigen. .Interestingly. CDw40. a putative B-cell activation antigen, shows home sequence homology with the NVGFR.“‘,” Cross reactivity, of anti-N(GFR with this activation antigen did not hkelv occur in our study since no lymphoid cells in the reactive or neoplastic cases marked with ;tnti-NGFR in frozen or paraf~n-er7lbedded tissue. 483
HUMAN PATHOLOGY
TABLE I.
Volume 22, No. 5 (May 1991)
Nerve Growth Factor Receptor Expression on Dendritic Reticulum Cells in Routinely Processed Lymph Nodes
Histologic Classification Reactive Follicular hyperplasia Lymphoma Follicular small cleaved cell type Follicular mixed cell type Follicular large cell type Small lymphocytic lymphoma Mantle zone lymphoma Hodgkin’s disease
Grade* 0
If
1
7
4 3
2+
2 -
10
1
6
3+
I?
more information regarding the diagnostic usefulness of such staining. The intimate association of DRCs with the neoplastic B cells in follicular lymphoma is particularly intriguing. The possible role of DRCs in the pathogenesis of follicular lymphoma may provide a fruitful area for future investigation.
No. Positive
25126
-
-
216
-
-
o/3
-
-
l/l 1
4
-
-
-
o/4
4 8
_ _
_
_
o/4 O/8
Acknowledgment.
The
NGFR-5
was
courteously
vided by A. Ross, PhD, Worcester Foundation mental Biology, Shrewsbury, MA.
pro-
for Experi-
REFERENCES 1. Jaffe ES: Histiocytoses of lymph nodes: Biology and differdiagnosis. Semin Diagn Pathol 5:376-390. 1988 2. Szakal AR: Microanatomy of lymphoid tissue during humoral immune responses. Annu Rev Immunol 7:91-109, 1989 3. Weisenburger DD. Harrington DS, Armitage JO: B-cell neoplasia. A conceptual understanding based on the normal humoral immune response. Pathol Annu 25:99-l 15. 1990 4. Naiem M, Gerdes J, Abdulaziz Z. et al: Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue. J Clin Path01 36:167-175. 1983 5. Carbone A, Manconi R, Poletti A, et al: Heterogeneous immunostaining patterns of follicular dendritic reticulum cells in human lymphoid tissue with selected antibodies reactive with different cell lineages. HUM PATHOL 19:51-56, 1988 6. Carbone A, Poletti A, Manconi R, et al: Heterogeneous in situ immunophenotyping of follicular dendritic reticulum cells in malignant lymphomas of B-cell origin. Cancer 60:2919-2926. 1987 7. Gloghini A, Colombatti A, Bressan G. et al: Basement membrane components in lymphoid follicles: Immunohistochemical demonstration and relationship to the follicular dendritic cell network. HUM PATHOL 20:1001-1007. 1989 8. Thompson SJ, Schatteman CC. Gown AM, et al: A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue. Am J Chn Pathol 92:415-423, 1989 9. Marano N. Dietzschold B, Earley JJ, et al: Purification and amino terminal sequencing of human melanoma nerve growth factor receptor. J Neurochem 48:225-232. 1987 10. Ross AH, Crab P. Bothwell M. et al: Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies. Proc Nat1 Acad Sci USA 8 1:6681-6685,1984 II. Chao MV. Bothwell MA, Ross AH, et al: Gene transfer and molecular cloning of the human NCF receptor. Science 232:518-521. 1986 ential
* Grading of staining patterns: 0, no dendritic staining detectable; 1 + , weak or focal dendritic staining; 2 + , focal intense or diffuse weak dendritic staining; 3+. diffuse, intense dendritic staining. Results of various follicles in each lymph node were av-
eraged.
generalized down-regulation of DRCs in the follicular lymphomas. Ultrastructural evidence of functional attenuation of DRCs in follicular lymphoma has also been previously reported. 24 Thus, DRCs may play a role in the pathogenesis of follicular lymphomas by virtue of their inability to regulate or inhibit the proliferation of the neoplastic B cells. However, further studies are needed to confirm this hypothesis. Four cases each of SL and MZL were examined. In SL, no staining of pseudofollicular proliferation centers was observed, indicating either an absence of DRCs within pseudofollicular proliferation centers or a loss of NGFRs on DRCs which might be present. Our recent study of SL with anti-DRC- 1 suggests that there are no DRCs in pseudofollicular proliferation centers.25 In all four cases of MZL, reactivity of the benign residual germinal centers was observed. These results are consistent with previous conclusions that the neoplastic cells in MZL originate from normal mantle zone lymphocytes rather than germinal center cells.26 The use of anti-NGFR immunohistochemistry for the discrimination of follicular hyperplasia from follicular lymphoma should be approached with caution. The number of cases examined in this study is small, and staining appears to be significantly affected by the method and quality of fixation. However, the application of this marker in selected cases in which the distinction between benign and neoplastic germinal centers is unclear may be helpful, especially when intense dendritic reactivity is present, favoring a reactive process. In conclusion, additional studies of NGFR expression in reactive and neoplastic follicular proliferations are warranted and may further elucidate the role of DRCs in these processes, as well as provide
12. Schatteman CC, Gibbs L, Lanahan AA, et al: Expression of NCF receptor in the developing and adult primate central nervous system. J Neurosci 8:860-873, 1988 13. Garin Chesa P, Rettig WJ. Thomson TM, et al: Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues. .J Histochem Cytochem 36:3X3-389, 1988 14. Perosio PM, Brooks JJ: Expression of nerve growth factor receptor in paraffin-embedded soft tissue tumors. Am J Clin Path01 132:152-160, 1988 15. Alavaikko M, Aine R, Rinne A, et al: Behaviour of dendritic reticulum cells possessing immunoreactive acid cysteineproteinase inhibitor in human lymphoid secondary follicles and in follicular-centre cell lymphomas. Int J Cancer 35:319-325, 1985 16. Gerdes 1. Stein H. Mason DY, et al: Human dendritic reticulum cells of lymphoid follicles: Their antigenic profile and their identification as multinucleated giant cells. Virchows Arch .IS1_ 42: 161-172, 1983 LZ
17.Braylan KC. Rappaport nodular lymphomas as compared sias. Blood 42:579-589, 1973
484
H: Tissue immunoglobulins in with reactive folhcular hyperpla-
NGFR EXPRESSION ON DRCs (Strobach et al)
generalized 1986
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lymphadenopathy
and
AIDS.
Am J Pathol
123:9-15,
?3. Alavaikko Yf. Rinne A, Jarvinen M. et al: Damage to secondary lymphoid follicles in AIDS-related persistent generalized lymphadenopathy: as revealed by the behaviour of dendritic reticulum cells possessmg immunoreactive acid cvsteine-proteinase inhibitor. Virchows Arch [B] 51):299-31 1, 19% 24. Rademakers LHPM. Peters JPJ, \an Llnnik JAM: Histiocvtir and dendritic reticulum cells in follicular structures of follicular lymphoma and reactive hyperplasia. Virrhows Arch [B] 44:X598, lYH3 25. O’<:onnor SJ, Dale); DT. U’eisenburger DD: Pseudofollicular proliferation centers in small lymphocvtic Iymphoma lack dendritic reticulum cells. Mod Pathol 2:67.4. 1989 (ahstr) 26. Weisenburger DD: Mantle zone Ivmphoma. histologic study. Cancer 53: 1073-1080. 1983
485
An immuno-