L a b o r a tor y in ves ti ga tio n Histopathology and immunohistochemist,y of dermatoborrelioses K E Y WORDS Lyme borreliosis, Borrelia burgdorferi, , dermato- · borrelioses, erythema migrans, borrelial lympho- cytoma, acrodermatitis chronica atrophicans, histo- pathology, light microscopy, immunohis- tochemistry Histopatholngy and immu,whistocliemistry of dermatoborrelioses W. Weger and R.R. Mtillegger SUMM ARY There are three different dermatological manifestations of LB (dermatoborrelioses, DB), erythema rnigrans (EM), borrelial lymphocytoma (BL), and acrodermatitis chronica atrophicans (ACA). The diagnosis of all three of these DB is primarily made on clinical grounds. Analysis of serum antibodies to B. burgdorferi is often unreliable and direct diagnostic methods (cultivation and PCR) are available in specialized labora- tories only. Histopathologic examination of biopsy samples from lesional skin of patients with suspected DB is a very helpful adjunct to the diagnosis. The most important finding in EM is a patchy mononuclear super- ficial (and deep); perivascular (and interstitial) infiltrate that is composed predominantly of lymphocytes and histiocytes with a variable admixture of plasma cells. There are two histopathologic types of BL, with (follicular type) or without (diffuse/nodular type) follicular structures, resembling the germinal cen- ters of lymph nodes. BL can be confused with well-differentiated nodular lymphoma histologically, but BL is always a pseudolymphoma, thus representing a benign lesion with a polyclonal proliferation of B and T cells. ACA develops clinically from an acute inflammatory phase into a chronic atrophic phase, which is reflected by different histopathologic features with a predominance of infiltrative changes in the early stage and cutaneous atrophy in the late stage. A significant finding in ACA is a patchy to band-like mononuclear infiltrate that is pronounced in the superficial dermis but also present in the deep portion of the dermis. The infiltrate is concentrated around blood vessels, which are often dilated. lmmunohis- tochemical investigation reveals in all forms of DB a mixed mononuclear infiltrate, in which CD8+ cells always outnumber CD4+ cells. Macrophages are found significantly more often in EM patients with as- sociated features than in those without. T cells are prevalent in EM, whereas B cells are prevalent in BL. Introduction Lyme borreliosis (LB), a nrnltisystemic infectious disease that is caused by the spirochete Borrelia burgdoiferi (Bb), bas a complex inflammatory pathol- ogy (1) that is reflectecl by clistinct histopathologic fea- tures at the site(s) of infection. There are three different dermatological manifestations of LB (dermatoborre- lioses, DE), erythema migrans (EM), borrelial lympho- cytoma (BL), and acrodermatitis chronica atrophicans Acta Dermatoven APA Vol 10, 2001, No 4 - ---------------------- - ------ -- JJJ Histopathology and immunohistochemist1y r!( dermaroborrelioses Laboratory investi gat ion Table. Expression of leukocyte differentiation antigens based on a score of 0-3 in lesional skin of patients with various manifestations of dermatoborrelioses (mean values ± standard cleviation) EM major 2.5 (±Q.7)"h 2.1 (±0.7)" O.S (±O.S) 2.5 (±O.S)' 1.0 (±0.6) EM minor 1.5 (±0.8) 2.3 (±Q.4)dc 0.6 (±0.4) 1.8 (±0.4)' 1.4 (±0.9) BL 2.5 (±0.8) 2.3 (±0.7) O.S (±O.O) 2.2 (±1.2)' 3.0 C±O.W ACA 2.2 (±0.6)" 1.8 (±0.9) 0.3 (±0.4) 1.6 (±0.9)' 2.Q (±Q.9)h a, p <0.05 vs CD20+ cells; b, p<0.05 vs EM minor; c, p=0.05 vs EM minor; d, p<0.05 vs CD20+ cells; e, p<0.05 vs CD68+ cells; f, p<0.05 vs CD4+ cells; g, p<0.05 vs EM major and minor; h, p=0.05 vs EM major (ACA). The diagnosis of ali three of these DB is prima- rily made on clinical grounds. Analysis of serum anti- boclies to Bb by ELISA tests is routinely performecl, but is supportive only in ACA, where a positive IgG titer is founcl in all patients, ancl in BL, where arouncl 80% of patients are seropositive. In EM, however, serologic cli- agnosis is very unreliable clue to frequent false positive or false negative results and lack of stanclarclization. Moreover, in Europe no single set of criteria for the in- terpretation of immunoblot analyses results in high lev- els of sensitivity and specificity (2). Direct diagnostic methocls inclucle cultivation ofBb from lesional skin of patients in a Barbour-Stoenner-Kelly meclium ancl cle- tection of Bb specific DNA by polymerase chain reac- tion (PCR). Cultivation of Eb may reach a sensitivity of 80% uncler optimal conditions, but is laborious ancl re- sults are not available before 1-4 weeks after biopsy clepencling on the multiplication ofthe spirochetes. PCR from skin biopsy samples is an aclvantageous technique as it amplifies the usually low amount of Eb specific DNA present, but is available in specializecl laborato- ries only ancl false positive results due to contamina- tion pose a potential problem. Thus, histopathologic examination of biopsy samples from lesional skin of patient, with suspectecl DE is a very helpful and straight- forward adjunct to the cliagnosis . The clinical diagnosis of DE can be supportecl ancl important differential cli- agnoses can be excluclecl primarily by the pattern and cellular composition ofthe inflammato1y infiltrate in the skin lesion. For histopathologic examination of DE, a 4-mm punch biopsy must be obtainecl uncler sterile conditions following local anesthesia from the leacling eclge of the rash in EM, from the central part of the nocl- ule or plaque in EL, and from the area with the most prominent signs of inflammation in ACA. The biopsy specimen has to be placecl immediately in 4% formal- clehycle/phosphate buffer saline ancl fixecl overnight, dehyclrated in graclecl ethanol ancl embeclcled in parat._ fin. Sections of 4 pm are then cut for histopathology (routine staining with hematoxylin ancl eosin), and of 5 pm for immunohistochemist1y. The present article describes the histopathologic ancl immunohistochemical features of the various manifes- tations of DB ancl reflects our continuing experience from more than 500 patient, biopsied at our department during the last eight years. Histopathology oj erythema migrans The most important finding in EM is a patchy mono- nuclear infiltrate of mile! to moderate or even severe density depending on the clinical clegree of inflamma- tion. The infiltrate is located mostly in the superficial but also in the deeper clermis with a perivascular con- centration, although there is also a less intense intersti- tial infiltrate (Figure 1). Periglanclular and perineural infiltrates may occur. The blood vessels arouncl which the infiltrates are accentuated may be clilated but are never clamagecl. The infiltrate is composecl preclomi- nantly of lymphocytes ancl histiocytes with a variable admixture of plasma cells. The number of plasma cells is usually greater in olcler lesions. In (ve1y) early lesions, a small nurnber of neutrophils ancl eosinophils may also be present (4), which can make the c.lifferentiation to unspecific tick or insect bite reactions c.lifficult. The early skin lesion arouncl a tick bite is characterizecl by an epidermal blister containing e1ythrocytes ancl nuclear clebris anc.l a superficial ancl cleep preclominantly neu- trophilic infiltrate, intermingled with lymphocytes, his- tiocytes, and eosinophils. This infiltrate is most intense 136 - - --------- - --- - - ------ - --- --- -----Acta Dermatoven APA Vol 10, 2001, No 4 L a b o r a rory invest i gar io n 1 2 Histopathology and immunohistochemistry of dermatoborrelioses Figure 1. Erythema migrans. Moderate to severe superficial and deep mostly perivascular mononuclear infiltrate. Hematoxylin and eosin, x10 Figure 2. Follicular type of borrelial lymphocytoma with a dense infiltrate predominantly in the deep dermis. Hematoxylin and eosin, x 1 O in the superficial part of the dermis and directly around the tick bite injury and is located around blood vessels as well as between collagen fibers. In adclition, edema of the papilla1y dermis, extravasation of erythrocytes , and vascular dilatation can be observed in tick bite le- sions (4-6) . Less important findings in EM, which are also not present in ali cases, are a slight edema of the papillary (and reticular) dermis, loss of pilosebaceous units, an increased number of fibroblasts/ fibrocytes , and fibro- sis of the reticular dermis. The epidermis in EM lesions is usually intact, but a thickened basket-weave horny layer, atrophy ancl/ or spongiotic foci may occur. Some- times a lymphocytic infiltrate is locatecl at the clermo- epiclermal junction with clisruption of the basement membrane (4) . Histopathology oj borrelial lymphocytoma BL is a lesion of cutaneous lymphoicl hyperplasia (7). There are two histopathologic types ofBL (5,6) w ith (follicular type) or without (cliffuse/ nodular type) folli- cular structures, resembling the germinal centers of lymph nodes (8). Combinations between the two types can be observecl. (i) In the cliffuse/ noclular type, a dense infiltrate of mature lymphocytes, lymphoicl cells with pale-staining inclentecl or cleaved nuclei, sometimes in clusters, plasma cells, and sometimes eosinophils ancl multinucleatecl giant cells can be observecl especially in the upper ancl mid dermis. There is a connective tis- sue background reaction with an increased number of fibroblasts , fibrosis, and edema. (ii) In the follicular type (Figure 2) , there is a clense, nodular infiltrate in the cleep clermis that may reach clown into the panniculus. This infiltrate forms follicu lar structures with germinal cen- ters, which consist of the same cells (polymorphic I ym- phoicl cells) as in the germinal centers of normal lym- phatic tissue, thus imitating secondary lymph node fol- licles. The infiltrate between the follicular structures is predominantly composed of small lymphocytes, plasma Acta Dermatoven APA Vol 10, 2001, No 4 -------- --------- --- ---- ------- - - 13 7 Histopathology and immunohistochemistry of dermatoborrelioses Figure 3. Acrodermatitis chronica atrophicans. Clustered plasma cells within a perivascular infiltrate. Hematoxylin and eosin, x 40 Figure 4. Acrodermatitis chronica atrophicans. Early lesion with dense patchy and subepidermal band-like infiltrate. Hematoxylin and eosin, x 1 O cells, some eosinophils , mast cells, and histiocytes. Edema and clilated vessels are seen in the papillary der- mis, w here the infiltrate is lacking. BL can be confused with well-differentiated nodu- lar lymphoma histologically, but BL is always a pseudo- lymphoma, thus representing a benign lesion with a polyclonal proliferation of Band T cells (4,9) . Impor- tant histopathologic differences to malignant lesions are a predominance of small lymphocytes, a mixed celi in- filtrate , and lack of necroses. In difficult situations (e .g. , predominance of lymphoid cells) it is necessary to prove polyclonality by immunohistochemistry (staining for Kappa and Lambda light chains) and PCR (T and B-cell rearrangement studies) . Histopathology oj acrodermatitis chronica atrophicans ACA develops clinically from an acute inflammatory phase into a chronic atrophic pluse over many weeks to months without adequate antibiotic treatment. This clinical course is reflected by different histopathologic features with a predominance of infiltrative changes in the early stage and cutaneous atrophy in the late stage of the disease. A significant finding in ACA is a patchy to band-like mononuclear infiltrate that is pronounced in the superficial dermis but also present in the deep portion of the dermis and sometimes reaches down into the pan niculus. The infiltrate is concentrated around blood vessels, which are often dilated, and skin append- ages, but also extends between collagen fibers. It is composed of lymphocytes and histiocytes, ancl (clus- tered) plasma cells (Figure 3) in greater numbers than in EM. The severity of the infiltrate is usually moderate to severe in early lesions (Figure 4) and mild to moder- ate in older lesions (Figure 5), but even in the chronic phase the infiltrate is stil! notable. Only in very late burned-out lesions , the infiltrate is sparse or may even be lacking (13). Atrophy of the epidermis with thinning and loss of rete ridges develops over tirne (Figure 5). In the late stage, the epidermis is often only a few celi layers thick. Interface dermatitis with necrosis of basal Lab o rator y inv estigation 138 - ---- - -----------------------------Acta Dermatoven APA Vol 10, 2001, No 4 Laboratory investigation 5 Histopathology and immunohistochemistty of dermatoborrelioses Figure 5. Acrodermatitis chronica atrophicans. Advanced lesion with mild superficial and deep dermal infiltrate and atrophy of the epidermis. Hematoxylin and eosin, x 1 O Figure 6. Fibrotic nodules over the elbow of a 45-year-old man. cells may be seen in those cases where the dermal infil- trate is lichenoid band-like. An increased number of fibroblasts and fibrosis may be explained by the fact that Bb is commonly aligned with collagen fibrils and activates fibroblasts through cytokines. Fibrosis starts to develop in the early phase of the disease. Degenera- tion ancl markecl recluction of collagen ancl elastic fi - bers, which accounts for a reduction of the breadth of the dermis, can be seen in the late stage. Elastic fibers are regularly surrounded by macrophages ancl multi- nucleated giant cells with elastophagocytosis, which may explain the loss of elastic fibers. This phenomenon can be observed on light microscopy (6) as well as on electron microscopy (11). Pilosebaceous units and sweat glands are first infiltrated ancl finally disappear as well. Another finding , which is less important, is an intersti- tial (mucinous) edema, which is often found in early lesions. Hauser (13) has clescribed small fat vacuoles and fat cells in ACA lesions, but Brehmer-Andersson et al. (12), who described these structures as small groups or massive bands of vacuoles in the upper half of the dermis, have suggested that they represent rather lympheclema or lymphostasis. Fibrotic nodules and sclerodermiform conditions in acrodermatitis chronica atrophicans Fibrotic (syn.: fibroicl, fibrous) nodules and/or pseudoscleroderma occur in 10% of ACA patients and are characterized by an increase in collagen in the der- mis. Fibrotic nodules are clome-shaped nodules with a hard consistency in juxtaarticular locations, such as over the clorsal aspects of the elbows (Figure 6) or knees. Fibrotic noclules can be confused with rheumatoid nod- ules and gouty tophi clinically, and histopathology is necessary for the distinction of these conclitions. The predominant finding is the presence of coarse, hyalini- zed collagen bundles (Figure 7) within the middle and cleep part of the broadenecl dermis, extending into the Acta Dermatoven APA Vol 10, 2001, No 4 ---------------------------------- 13 9 Histopathology and immunohistochemistry of dermatoborrelioses subcutaneous fat. The collagen fibers are arranged in peculiar anion-like concentric , or cartwheel-like , or in- terlacing patterns around a homogenous eosinophilic center. The number of fibroblasts is markedly increased within the entire dermis. A miki lympho-plasmacellular and histiocytic infiltrate , sometimes with eosinophils, is faund in the upper dermis around blood vessels and accompanies the collagen formations in bands. The density of the infiltrate decreases towards the center of the collagen formations. The overlying epidermis is usually atrophic with some orthokeratosis . There is a complete loss of adnexal structures. Bb is well culti- vable from fibrotic nodules (14) . Laboratory in vestigation Sclerodermifarm (morphea-like) conditions, named pseudoscleroderma, can be faund within ACA lesions , and appear clinically as whitish, ivory-colored indurated areas. Histopathologically, the epidermis is thinned and flattened in these areas. Homogenous hyalinized col- lagen is faund within the excessively broadened der- mis, and the number of fibroblasts, which entrap the collagen bundles, is markedly increased. Aggregates of lymphocytes and plasma cells are arranged around blood vessels but also interstitially. Eosinophils may be present within these infiltrates. The subcutaneous fat is largely replaced by collagen. Figure 7. Fibrotic nodule. Coarse interlacing collagen bundles with mild mononuclear infiltrate accompanying the collagen formations in bands. Hematoxylin and eosin, x 1 O Figure 8. Diffuse type of borrelial lymphocytoma. Moderate numbers of CD3+ T cells within perivascular infiltrates. lmmunoperoxidase stained with anti-CD3 antibodies. x 25 Immunohistochemistry oj dermatoborrelioses We have analyzed biopsy specimens from 6 EM pa- tients without extracutaneous signs and symptoms (EM minor) , 6 EM patients with associated features (EM major), 5 BL patients, and 10 ACA patients far the ex- pression of leukocyte differentiation antigens CD68 (macrophages), CD3 (T cells), CD4 (T-helper cells), CD8 (T-suppressor cells), and CD20 (B cells) using the avi- din-biotin-peroxidase complex method (15). The per- centage of cells positive far a respective leukocyte marker was estimated relative to the total number of infiltrating cells and positivity was graded on a scale from 0-3. The results were statistically comparecl be- tween the patient groups (Table). Among the 6 patients with EM major, great numbers of macrophages were fauncl; T cells were seen in moderate number, and B cells were infrequent. Among the 6 patients w ith EM minor, T cells were the preclominant celi type, ancl they were present in significantly higher numbers than mac- rophages or B cells. When the two groups were com- parecl, macrophages were cletectecl significantly more often among EM major patients. In BL lesions, most of , 8 140 ---------- ----------- --------------Acta Dermatoven APA Vol 10, 2001, No 4 Laboratory investigation Histopathology and immunohistochemist1y t/{ dermatoborrelioses the cells were B lymphocytes, but T celis (Figure 8) and macrophages were also present in significant numbers. In the 10 ACA patients, macrophages, T celis, and B celis were founcl in moderate numbers. CDS+ celis out- numbered CD4+ celis significantly in ali patient groups. Two other examinations of leukocyte surface markers in DB have been performecl so far. Biichner ancl Rufli (16) analyzed 9 patients with EM ancl found that the perivascular infiltrates within the erythematous periph- eral portion of EM were composed preclominantly of LEU4+ T celis (CD3). In contrast to our study, LEU3a+ helper cells (CD4) were more numerous than LEU2a+ suppressor T celis (CDS). The authors also found a high number ofLEU6+ Langerhans celis in the epidermis ancl dermis in the same areas. Brehmer-Andersson et al. (12) have examined 7 patients with ACA by PAP immuno- peroxiclase staining and have founcl that the lympho- cytes were T cells mainly expressing the UCHLl anti- gen (CD45RO), whereas ve1y few lymphocytes ex- pressecl B-cell antigen, clespite the presence of numer- ous plasma cells. Corresponding to this considerable number of plasma celis in ACA, we founcl approximately the same number of B cells and T cells in our patients. Conclusions There are integrative ancl clisintegrative histopatho- logic elements in DB. Common to ali manifestations of DB is the presence of a lymphohistiocytic infiltrate in- termingled with plasma celis (typical LB infiltrate) and a characteristic connective tissue reaction. Differences between the various manifestations of DB concern the pattern of the infiltrate, the proportion of t:he cell types within the infiltrate, the intensity of the infiltrate and the connective tissue reaction, and the presence or ab- sence of epidermal changes. The integrative immuno- histochemical elements in ali forms of DB are the pres- ence of a mixed mononuclear infilt:rate, in which CDS+ cells always outnumber CD4+ cells. Differences con- cern the number of macrophages, which are found sig- nificantly more often in patients with EM major than in those with EM minor. Furthermore, T cells are preva- lent in EM, whereas B cells are prevalent in BL. R 'l?l?'l<' I) l<'N, T(']!'.~ ._.1 .1. J .. .1 l . J .. .J .... _,, .. .4 S l. Steere AC. Lyme disease. N EnglJ Med 2001; 345: 115-25. 2. Robertson J, Guy E, Andrews N, Wilske B, Anda P, Granstrom M, et al. A European multicenter study of immunoblotting in serodiagnosis of Lyme borreliosis. J Ciin Microbiol 2000; 38: 2097-102. 3. Duray PH. Visceral histopathology in Lyme borreliosis: new observations. Zentralbl Bakteriol 1989; Suppl.18: 116-25. 4. De Koning J, Duray PH. Histopathology of human Lyme borreliosis. In: Weber K, Burgdorfer W ( edi- tors) . Aspects ofLyme borreliosis. 1993; Springer, Berlin-Heidelberg-New York; pp 70-92. 5. Hodl S, Soyer HP. Dermatopathology of Lyme borreliosis. Acta dermatovenerologica APA 1994; 3: 89-98. 6. Hodl S, Soyer HP, Mtillegger RR. Dermatopathologic diagnosis of Lyme borreliosis. Acta dermatovenerologica APA 1996; 5: 123-9. 7. Bafverstedt B. Uber Lymphadenosis benigna cutis. Acta Derm Venereol (Stockh) 1943; (Suppl.11)24: 1-202. 8. Lennert K, Stein H. The germinal center: morphology, histochemistry and immunohistology. In: Goos U, Christophers E (editors). Lymphoproliferative disease of the skin. 1982; Springer, Berlin-Heidelberg- New York. 9. Brady SP, Mtillegger RR, Steere AC, Wolfe HJ. Assessment of lymphoid clonality in acrodermatitis chronica atrophicans using moleq1lar analyses. Mod Pathol 1998; 11: 47A. 10. AsbrinkE, HovmarkA. Earlyand late cutaneous manifestations oflxodes-borne borreliosis (Erythema migrans borreliosis, Lyme borreliosis). Ann N Y Acad Sci 1988; 539: 4-15. 11. De KoningJ, Tazelaar DJ, Hoogkamp-Korstanje JAA, ElemaJD. Acrodermatitis chronica atrophicans: a light and electron microscopic study. J Cutan Pathol 1995; 22: 23-32. 12. Brehmer-Andersson E, Hovmark A, Asbrink E. Acrodermatitis chronica atrophicans: histopathologic findings and clinical correlations in 111 cases. Acta Derm Venereol (Stockh) 1998; 78: 207-13. 13. Hauser W. Wahrscheinliche Infektionskrankheiten der Haut. In: Jadassohn J ( editor). Handbuch der Haut- und Geschlechtskrankheiten IV, lA. 1965; Springer, Berlin-Heidelberg-New York, pp 556-629. Acta Dermatoven APA Vol 10, 2001, No 4 - - -------------------- - - - ------- 141 Histopathology and immunohistochemist,y