INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY
Vol. 27, no. 4, 669-674 (2014)
LETTER TO THE EDITOR
SEVERE SEPTAL PANNICULITIS IN A MULTIPLE SCLEROSIS PATIENT TREATED WITH INTERFERON-BETA E. MAZZONI, C. GUARNERF, S. GIACOPPOI, C. RIFICP, G. TCHERNEV3, G. POLIMENI4 and U. WOLLINA5
Experimental Neurology Laboratory, IRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy; 'Department ofClinical Experimental Medicine, University ofMessina, Messina, Italy; 3Policlinic for Dermatology and Venerology, Saint KlimentOhridski University, Sofia, Bulgaria; "Department ofClinical Experimental Medicine, University ofMessina, Messina, Italy; 5Department ofDermatology and Allergology, Academic Teaching Hospital ofthe Technical University ofDresden, Dresden, Germany
1
Received August 28, 2014 - Accepted October 13, 2014 We report a memorable case of severe septal panniculitis in an MS patient following the subcutaneous administration of interferon beta-lb, manifesting as a painful, indurated, erythematous lesion of the thigh, which appeared at the injection site. 8 million IV subcutaneously every other day into the abdomen and thighs. After eight years of therapy he developed a painful, erythematous swelling and tender induration at the site of injection on his left thigh. Mild fever and headache were also present. The results of laboratory tests, including blood count and chemistry profile were within normal limits. There was no personal or family history of thromboembolic disease. He was empirically given amoxicillin plus clavulanate for ten days for suspected cellulitis, with no significant improvement of the skin condition. Incision and drainage was attempted, but yielded no pus. Culture of swabs for bacteria was positive only for resident flora, no fungi. Histological analysis of a biopsy specimen showed thrombosis of the vessels of the dermis and an important inflammatory infiltration with greater localization in the fat lobules and the septa (Fig. 1). As a result, the MS patient was diagnosed with
Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system that is characterized by inflammation, demyelination and neurodegeneration within the brain and the spinal cord. Treatment of MS is currently limited to reducing disease progression via disease-modifying treatments that modulate the immune system, administered intramuscularly or subcutaneously. Skin reactions associated with these drugs are common and may variously influence the patient's health-related quality of life. In this setting, severe local skin reactions to subcutaneous injection of interferon beta-lb are rare, and only 14 cases have been reported to date. Case report
Recombinant human interferon beta-I b therapy was begun in 2005 on a 44-year-old man with an eleven-year history of MS. The patient self-injected
Key words: severe septal panniculitis, multiple sclerosis, interferon beta-I b Mailing address: Claudio Guarneri, M.D Department of Clinical and Experimental Medicine, University of Messina, Messina (Italy) Policlinico Universitario "G. Martino", via Consolare Valeria Gazzi, 98125 Messina, Italy Tel.: +390902212894 Fax: +390902927691 e-mail: [email protected]
0394-6320 (2014)
669
Copyright © by BIOLIFE, s.a.s. This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO TIDS ARTICLE.
E. MAZOZN ET AL.
670
severe septal panniculitis secondary to interferon beta-l b treatment. In detail, panniculitis is defined as an inflammation of the layer of subcutaneous fat underlying the epidermis of the skin (1), caused by a wide range of factors and diseases, such as infections, systemic diseases, trauma, drugs and others, but in many cases the cause remains unknown. Panniculitis is classified based on the part of the fatty tissue involved and the involvement of blood vessels in the inflamed area (vasculitis). In septal panniculitis the inflammation is centred upon the connective tissue septa. There is often some spillover of the inflammatory cells into the periphery of the lobule (2). Furthermore, immunohistochemical analysis of the inflammatory cells shows positive staining for CD4 and for CD8 alpha (both expressed in T helper cells and cytotoxic cells), whereas immunohistochemical localization for chymase was negative. The expression of proinflammatory cytokines such as TNF-n was also analyzed, and positive staining was localized mainly in the infiltrated inflammatory cells in damaged tissues (Fig. 2). The positivity for TNF-n correlated with an intense signal immunolocalization for I-CAMl. MATERIALS AND METHODS Light microscopy Tissue biopsy, taken from the lesion on the patient's thigh, was fixed in 10% (w/v) PBS-buffered formaldehyde, Tissue sections were first paraffin-embedded and cut into 7 urn-thick sections and then were deparaffinized with xylene, stained with haematoxylin/eosin (H&E) and studied using light microscopy (LEICA ICC50 HD microscope). Immunohistochemical localization for CD4, CD8-a,. ICAM-l, TNF-a and chymase Sections prepared from paraffin-embedded tissues were deparaffinated and endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with anti-CD4 polyclonal antibody (Santa Cruz Biotechnology, I: 100 in PBS); anti-CD8-a polyclonal antibody (Santa Cruz
Biotechnology, 1:100 in PBS); anti-ICAM-l monoclonal antibody (Santa Cruz Biotechnology, I: 100 in PBS), antiTNF-a polyclonal antibody (Cell Signaling Technology, I: 100 in PBS) and anti-Chymase monoclonal antibody (Millipore, I :250 in PBS). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat antirabbit IgG and avidin-biotin peroxidase complex (DBA). The counterstain was developed with hematoxylin (blue background) and avidin-biotin peroxidase (brown staining). A positive staining (brown) was found in the sections, indicating that the immunoreactions were positive. To verify the binding specificity, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out. All images were acquired in light microscopy using a LEICA ICC50 HD microscope.
RESULTS Following histological analysis of a biopsy specimen of thigh lesions, which appeared at the interferon beta-lb injection site, diagnosis of severe septal panniculitis was made for the MS patient. The lesion was further treated with fusidic acid ointment and the pain and induration markedly improved within three weeks; a small ulcerative lesion in the center of the affected area remained for five months, showing periods of epithelialization and re-ulceration, then leaving a depressed scar. In accordance with the patient's wishes, recombinant interferon beta-lb therapy was discontinued. With recrudescence of MS signs and symptoms, the patient was recommenced on therapy after proposing a new automated injection device, with training on the appropriate deep selfadministration technique, avoiding the site of the previous lesion. The patient has not developed any further skin reactions. DISCUSSION MS is a chronic, immune-mediated inflammatory disease of the central nervous system, which is characterized by infiltration of immune cells, loss of myelin and axons, and formation of multifocal plaques in the brain and the spinal cord (3). It is a
Int. J. Immunopatbol. Pbarmacol.
671
Fig. 1. Haematoxylin and eosin stain (H&E). Histological alterations were evaluated on the patient 's thigh lesions and stain ed with H&E.
A
c
D
Fig. 2. Immunohistochemical analysis. Positive staining was observedfor CD4 (A), CD8-a (B), ICAM-I (C) and TNF-a (D) in tissue sections, while has not been demonstrated po sitive stainingfor Chymase (E) .
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E. MAZOZN ET AL.
relatively common disease, with approximately 2.5 million people being affected worldwide, most of whom are young or middle-aged adults (4). As a definitive cure is still lacking, treatment options are limited to modulate the immune system, thus reducing symptoms and disease progression (5). In this setting, disease modifying therapies (DMTs) actually include Glatiramer acetate and interferon beta-la and interferon beta-lb, both affecting multiple immunological processes (6). They require subcutaneous administration although interferon beta-l b la can be injected intramuscularly. Both therapies are generally safe and well tolerated, with an acceptable side-effect profile. However, apart from general symptoms (f1.ulike syndrome, headache, increased spasticity, anaphylactic shock and psychological changes) there are several reports in literature on possible cutaneous adverse events in using these drugs (7). Local skin reactions to subcutaneous injections of interferonbeta-l b in MS include a wide range of differently recognized clinical manifestations, varying from redness/erythema, swelling, induration, to necrosis, accompanied by pruritus, inflammation! irritation, bruising to burning or pain at the site of injections and surrounding area (8, 9). Psoriatic eruptions have also been described (10, 11). Here, we report the case of a 44-year-old male diagnosed with MS, who developed painful, erythematous swelling and tender induration at the site of injection on his left thigh. With this case report, we would like to draw attention to severe septal panniculitis, a serious drug complication, that develops following interferon beta-lb treatment after an improperly administered injection. Although severe septal panniculitis at the injection sites is a rare event, it has a relevant impact on the patient's quality of life during interferon therapy. As reported in the clinical trials (7), the proportion of patients with MS treated with interferon beta1b presenting such cutaneous reactions is between 13% and 89%, being mostly females with low-mild grade of severity in skin disease (12), that resolves spontaneously. Severe local skin reactions have rarely been reported, only 14 cases in literature, leading to some problems in diagnosis and management of these lesions, together with a loss of patient's compliance
and discontinuation ofMS treatment (6, 13, 14). Patients are mainly middle-aged (mean 42.9 years) women (78.6%) (15). Many cases had multiple lesions, located on typical sites of subcutaneous injection, represented by erythematous indurations with possible features of necrosis in the center usuallydeveloping in a second time (6, 16). The exact reasons underlying these reactions are still debated. Similar manifestations have been reported with subcutaneous injections of interferon beta-l a, suggesting that the same mechanisms involving receptor sites, immunological and proinflammatory properties of both interferons are likely to be identified as potential triggers of these phenomena (17, 18). Indeed, recently, the case was reported of a 44-year-old diagnosed with MS who developed septal panniculitis following interferon beta-la treatment after an improperly administered intramuscular injection (19). Also, in literature the case is described of a 43-year-old woman affected by MS, who self-administered interferon beta-la subcutaneously 3 times a week. After 18 months of treatment, patient was diagnosed with mixed panniculitis secondary to therapy (20). Therefore, it is noteworthy that development of septal panniculitis may be the result of treatment with other systemic drugs, such as interferon beta-la, not only interferon beta-lb. Some authors also speculated on the fact that such reactions occur even more frequently in association with IFN-~-lb compared to IFN-~-la. Immunologicall- mediated necrotizing vasculitis and platelet-dependent thrombosis in the dermis may play a role. Multiple sclerosis per se or its immunomodulatory treatment seems to trigger clotting abnormalities, as shown by a consistent activation of plateles in these patients (21). Moreover, authors demonstrated the direct induction of local chemokine expression and the associated immune cell extravasation caused by interferon beta in human skin biopsy specimen (22). Histologically, perivascular dermatitis or panniculitis have been observed, with or without thrombosisofthe dermal vessels and leukocytoclastic vasculitis with perivascular fibrinoid material and nuclear dust in some cases. Lymphocytes are the main cell population (7, 15,23). The release of inflammatory cytokines from
Int. J. Immunopathol. Pharmacol.
subcutaneous adipose tissue even after minimal trauma, as also proved in our case by the positivity of TNF-(1 staining, mainly in, the infiltrated inflammatory cells in damaged tissues, may play a role in the inflammatory response (24). Also, the positivity for TNF-(1 correlated with an intense signal immunolocalization for I-CAM1, in accordance with previous studies, showing that TNF-(1 induces the upregulation of ICAM-l (25, 26). ICAM-l is an adhesion molecule expressed in leukocytes and endothelial cells, which is involved in recruitment of inflammatory cells, such as leukocytes and lymphocytes (27). Also, mast cells are implicated in leukocyte recruitment and tissue damage, indeed histamine is released through their degranulation. Moreover, immunohistochemical localization for chymase was performed to detect the presence of mast cells, but the section did not stain for this marker, thus confirming that allergic reaction is not involved in the pathogenesis of these lesions. On the contrary, positive staining was found by immunohistochemical analysis for CD4 and for CD8 alpha, which play a key role in cell-mediated immunity. In agreement with other studies, this work should enhance cognition that severe septal panniculitiscan also occurs as an adverse effect, although rarely, following subcutaneous injection with interferon beta-lb. Thus, we suggest that early recognition and correction of the injection technique may help to prevent severe complications. Moreover,authorsunderlinetherole ofintradermal injection as possible trigger of the adverse immune reaction, suggesting deep cutaneous penetration of the drug to avoid these reactions; in fact, the use of adequate training and/or automated injection devices has .been associated with an estimated 60% rate of reduction in the incidence of adverse cutaneous side effects (12,17,24,28). Certainly, discontinuation of the drug is followed by a rapid recrudescence of the disease, so education of MS patients regarding the correct administration of DMTs is mandatory in daily management of this invalidating condition (24, 28). At the same time, physicians should be familiar with these lesions for prompt diagnosis and treatment of both cutaneous and neurological diseases.
673 REFERENCES
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Johnson MA, Kannan DG, Balachandar TG, Jeswanth S, Rajendran S, Surendran R. Acute septal panniculitis. A cutaneous marker of a very early stage of pancreatic panniculitis indicating acute pancreatitis. JOP 2005; 6:334-8. 2. Calonje E. Dermatopathology: LC07-l PANNICULITIS. Pathology 2014; 46(S)2:S10. 3. Frohman EM, Racke MK, Raine CS. Multiple sclerosis-the plaque and its pathogenesis. NEngl J Med 2006; 354:942-55. 4. Johnston J, So TY. First-line disease-modifying therapies in paediatric multiple -solerosis: a comprehensive overview. Drugs 2012; 72:1195-211. 5. Chun J, Brinkmann V. A mechanistically novel, first oral therapy for multiple sclerosis: the development offingolimod (FTY720, Gilenya). Discov Med 2011; 12:213-28. 6. Weise G, Hupp M, Kerstan A, Buttmann M. Lobular panniculitis and lipoatrophy of the thighs with interferon-ss1a for intramuscular injection in a patient with multiple sclerosis. J Clin Neurosci 2012; 19:1312-3. 7. Balak DM, Hengstman GJ, Cakmak A, Thio HB. Cutaneous adverse events associated with diseasemodifying treatment in multiple sclerosis: a systematic review. Mult Scler 2012; 18:1705-17. 8. Giovannoni G, Barbarash 0, Casset-Semanaz F, et al. Safety and immunogenicity of a new formulation of interferon beta-la (RebifNew Formulation) in a Phase IIIb study in patients with relapsing multiple sclerosis: 96-week results. Mult Scler 2009; 15:21928. 9. Inafuku H, Kasem Khan MA, Nagata T, Nonaka S. Cutaneous ulcerations following subcutaneous interferon beta injection to a patient with multiple sclerosis. J Dermatol 2004; 31:671-7. 10. Webster GF, Knobler RL, Lublin FD, Kramer EM, Hochman LR. Cutaneous ulcerations and pustular psoriasis flare caused by recombinant interferon beta injections in patients with multiple sclerosis. J Am Acad Dermatol1996; 34:365-7. 11. Kowalzick L. Psoriasis flare caused by recombinant interferon beta injections. J Am Acad Dermatol
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1997; 36:501. 12. Lebrun C, Bertagna M, Cohen M. Cutaneous sideeffects ofimmunomodulators in MS. Int MS J 2011; 17:88-94. 13. Ball NJ, Cowan BJ, Hashimoto SA. Lobular panniculitis at the site of subcutaneous interferon beta injections for the treatment of multiple sclerosis can histologically mimic pancreatic panniculitis. A study of 12 cases. J Cutan Pathol 2009; 36:331-7. 14. Heinzerling L, Dummer R, Burg G, SchmidGrendelmeier P. Panniculitis after subcutaneous injection of interferon beta in a multiple sclerosis patient. Eur J Dermatol2002; 12:194-7. 15. Nakamura Y, Kawachi Y, Furuta J, Otsuka F. Severe local skin reactions to interferon beta-l b in multiple sclerosis-improvement by deep subcutaneous injection. Eur J Dermato12008; 18:579-82. 16. O'Sullivan SS, Cronin EM, Sweeney BJ, Bourke JF, Fitzgibbon 1. Panniculitis and lipoatrophy after subcutaneous injection of interferon beta-l b in a patient with multiple sclerosis. J Neurol Neurosurg Psychiatry 2006; 77:1382-3. 17. Balak DM, Hengstman GJ, Hajdarbegovic E, van den Brule RJ, Hupperts RM, Thio HB. Prevalence of cutaneous adverse events associated with long-term disease-modifying therapy and their impact on healthrelated quality oflife in patients with multiple sclerosis: a cross-sectional study. BMC Neuro12013; 13:146. 18. Soria A, Maubec E, Henry-Feugeas MC, et al. Panniculitis induced by interferon beta-la vascular toxicity. Ann Dermatol Venereo12007; 134:374-7. 19. Ozuguz P, Kacar SD, Karaca S, Tokyol C. A case of septal panniculitis secondary to interferon treatment. Cutan Ocul Toxicol. 2014 Mar 18. [Epub ahead of print] 20. Cuesta L, Moragon M, Perez-Crespo M, Onrubia J,
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Garcia M. Mixed panniculitis secondary to interferon beta-la therapy in a woman with multiple sclerosis. Aetas Dermosifiliogr 2013; 104:257-9. Ozden MG, Erel A, Erdem 0, Oztas MO. Dermal fibrosis and cutaneous necrosis after recombinant interferon beta la injection in a multiple sclerosis patient. J Eur Acad Dermatol Venereo12005; 19:11213. Poulin F, Rico P, Cote J, Begin LR. Interferon betainduced panniculitis mimicking acute appendicitis. Arch Dermato12009; 145:916-77. Elgart GW, Sheremata W, Abo YS. Cutaneous reactions to recombinant human interferon beta1b: the clinical and histologic spectrum. J Am Acad Dermatol1997; 37:553-8. Pachler C, Ikeoka D, Plank J, et al. Subcutaneous adipose tissue exerts proinflammatory cytokines after minimal trauma in humans. Am J Physiol Endocrinol Metab 2007; 293:E690-6. Chen C, Chou C, Sun Y; Huang W. Tumor necrosis factor alpha-induced activation of downstream NFkappaB site of the promoter mediates epithelial ICAM-l expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway. Cell Signal 2001; 13:543-53. Yang L, Froio RM, Sciuto TE, Dvorak AM, Alon R, Luscinskas FW. ICAM-l regulates neutrophil adhesion and transcellular migration of TNF-alphaactivated vascular endothelium under flow. Blood 2005; 106:584-92. Frank PG, Lisanti MP. ICAM-l: role in inflammation and in the regulation of vascular permeability. Am J Physiol Heart Circ Physio12008; 295:H926-H927. Wollina U. Glatiramer acetate-induced lipoatrophy. Kosmetiche Medezin 2006; 27: 174-75.
Vol. 27, no. 4, 669-674 (2014)
LETTER TO THE EDITOR
SEVERE SEPTAL PANNICULITIS IN A MULTIPLE SCLEROSIS PATIENT TREATED WITH INTERFERON-BETA E. MAZZONI, C. GUARNERF, S. GIACOPPOI, C. RIFICP, G. TCHERNEV3, G. POLIMENI4 and U. WOLLINA5
Experimental Neurology Laboratory, IRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy; 'Department ofClinical Experimental Medicine, University ofMessina, Messina, Italy; 3Policlinic for Dermatology and Venerology, Saint KlimentOhridski University, Sofia, Bulgaria; "Department ofClinical Experimental Medicine, University ofMessina, Messina, Italy; 5Department ofDermatology and Allergology, Academic Teaching Hospital ofthe Technical University ofDresden, Dresden, Germany
1
Received August 28, 2014 - Accepted October 13, 2014 We report a memorable case of severe septal panniculitis in an MS patient following the subcutaneous administration of interferon beta-lb, manifesting as a painful, indurated, erythematous lesion of the thigh, which appeared at the injection site. 8 million IV subcutaneously every other day into the abdomen and thighs. After eight years of therapy he developed a painful, erythematous swelling and tender induration at the site of injection on his left thigh. Mild fever and headache were also present. The results of laboratory tests, including blood count and chemistry profile were within normal limits. There was no personal or family history of thromboembolic disease. He was empirically given amoxicillin plus clavulanate for ten days for suspected cellulitis, with no significant improvement of the skin condition. Incision and drainage was attempted, but yielded no pus. Culture of swabs for bacteria was positive only for resident flora, no fungi. Histological analysis of a biopsy specimen showed thrombosis of the vessels of the dermis and an important inflammatory infiltration with greater localization in the fat lobules and the septa (Fig. 1). As a result, the MS patient was diagnosed with
Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system that is characterized by inflammation, demyelination and neurodegeneration within the brain and the spinal cord. Treatment of MS is currently limited to reducing disease progression via disease-modifying treatments that modulate the immune system, administered intramuscularly or subcutaneously. Skin reactions associated with these drugs are common and may variously influence the patient's health-related quality of life. In this setting, severe local skin reactions to subcutaneous injection of interferon beta-lb are rare, and only 14 cases have been reported to date. Case report
Recombinant human interferon beta-I b therapy was begun in 2005 on a 44-year-old man with an eleven-year history of MS. The patient self-injected
Key words: severe septal panniculitis, multiple sclerosis, interferon beta-I b Mailing address: Claudio Guarneri, M.D Department of Clinical and Experimental Medicine, University of Messina, Messina (Italy) Policlinico Universitario "G. Martino", via Consolare Valeria Gazzi, 98125 Messina, Italy Tel.: +390902212894 Fax: +390902927691 e-mail: [email protected]
0394-6320 (2014)
669
Copyright © by BIOLIFE, s.a.s. This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO TIDS ARTICLE.
E. MAZOZN ET AL.
670
severe septal panniculitis secondary to interferon beta-l b treatment. In detail, panniculitis is defined as an inflammation of the layer of subcutaneous fat underlying the epidermis of the skin (1), caused by a wide range of factors and diseases, such as infections, systemic diseases, trauma, drugs and others, but in many cases the cause remains unknown. Panniculitis is classified based on the part of the fatty tissue involved and the involvement of blood vessels in the inflamed area (vasculitis). In septal panniculitis the inflammation is centred upon the connective tissue septa. There is often some spillover of the inflammatory cells into the periphery of the lobule (2). Furthermore, immunohistochemical analysis of the inflammatory cells shows positive staining for CD4 and for CD8 alpha (both expressed in T helper cells and cytotoxic cells), whereas immunohistochemical localization for chymase was negative. The expression of proinflammatory cytokines such as TNF-n was also analyzed, and positive staining was localized mainly in the infiltrated inflammatory cells in damaged tissues (Fig. 2). The positivity for TNF-n correlated with an intense signal immunolocalization for I-CAMl. MATERIALS AND METHODS Light microscopy Tissue biopsy, taken from the lesion on the patient's thigh, was fixed in 10% (w/v) PBS-buffered formaldehyde, Tissue sections were first paraffin-embedded and cut into 7 urn-thick sections and then were deparaffinized with xylene, stained with haematoxylin/eosin (H&E) and studied using light microscopy (LEICA ICC50 HD microscope). Immunohistochemical localization for CD4, CD8-a,. ICAM-l, TNF-a and chymase Sections prepared from paraffin-embedded tissues were deparaffinated and endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with anti-CD4 polyclonal antibody (Santa Cruz Biotechnology, I: 100 in PBS); anti-CD8-a polyclonal antibody (Santa Cruz
Biotechnology, 1:100 in PBS); anti-ICAM-l monoclonal antibody (Santa Cruz Biotechnology, I: 100 in PBS), antiTNF-a polyclonal antibody (Cell Signaling Technology, I: 100 in PBS) and anti-Chymase monoclonal antibody (Millipore, I :250 in PBS). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat antirabbit IgG and avidin-biotin peroxidase complex (DBA). The counterstain was developed with hematoxylin (blue background) and avidin-biotin peroxidase (brown staining). A positive staining (brown) was found in the sections, indicating that the immunoreactions were positive. To verify the binding specificity, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out. All images were acquired in light microscopy using a LEICA ICC50 HD microscope.
RESULTS Following histological analysis of a biopsy specimen of thigh lesions, which appeared at the interferon beta-lb injection site, diagnosis of severe septal panniculitis was made for the MS patient. The lesion was further treated with fusidic acid ointment and the pain and induration markedly improved within three weeks; a small ulcerative lesion in the center of the affected area remained for five months, showing periods of epithelialization and re-ulceration, then leaving a depressed scar. In accordance with the patient's wishes, recombinant interferon beta-lb therapy was discontinued. With recrudescence of MS signs and symptoms, the patient was recommenced on therapy after proposing a new automated injection device, with training on the appropriate deep selfadministration technique, avoiding the site of the previous lesion. The patient has not developed any further skin reactions. DISCUSSION MS is a chronic, immune-mediated inflammatory disease of the central nervous system, which is characterized by infiltration of immune cells, loss of myelin and axons, and formation of multifocal plaques in the brain and the spinal cord (3). It is a
Int. J. Immunopatbol. Pbarmacol.
671
Fig. 1. Haematoxylin and eosin stain (H&E). Histological alterations were evaluated on the patient 's thigh lesions and stain ed with H&E.
A
c
D
Fig. 2. Immunohistochemical analysis. Positive staining was observedfor CD4 (A), CD8-a (B), ICAM-I (C) and TNF-a (D) in tissue sections, while has not been demonstrated po sitive stainingfor Chymase (E) .
672
E. MAZOZN ET AL.
relatively common disease, with approximately 2.5 million people being affected worldwide, most of whom are young or middle-aged adults (4). As a definitive cure is still lacking, treatment options are limited to modulate the immune system, thus reducing symptoms and disease progression (5). In this setting, disease modifying therapies (DMTs) actually include Glatiramer acetate and interferon beta-la and interferon beta-lb, both affecting multiple immunological processes (6). They require subcutaneous administration although interferon beta-l b la can be injected intramuscularly. Both therapies are generally safe and well tolerated, with an acceptable side-effect profile. However, apart from general symptoms (f1.ulike syndrome, headache, increased spasticity, anaphylactic shock and psychological changes) there are several reports in literature on possible cutaneous adverse events in using these drugs (7). Local skin reactions to subcutaneous injections of interferonbeta-l b in MS include a wide range of differently recognized clinical manifestations, varying from redness/erythema, swelling, induration, to necrosis, accompanied by pruritus, inflammation! irritation, bruising to burning or pain at the site of injections and surrounding area (8, 9). Psoriatic eruptions have also been described (10, 11). Here, we report the case of a 44-year-old male diagnosed with MS, who developed painful, erythematous swelling and tender induration at the site of injection on his left thigh. With this case report, we would like to draw attention to severe septal panniculitis, a serious drug complication, that develops following interferon beta-lb treatment after an improperly administered injection. Although severe septal panniculitis at the injection sites is a rare event, it has a relevant impact on the patient's quality of life during interferon therapy. As reported in the clinical trials (7), the proportion of patients with MS treated with interferon beta1b presenting such cutaneous reactions is between 13% and 89%, being mostly females with low-mild grade of severity in skin disease (12), that resolves spontaneously. Severe local skin reactions have rarely been reported, only 14 cases in literature, leading to some problems in diagnosis and management of these lesions, together with a loss of patient's compliance
and discontinuation ofMS treatment (6, 13, 14). Patients are mainly middle-aged (mean 42.9 years) women (78.6%) (15). Many cases had multiple lesions, located on typical sites of subcutaneous injection, represented by erythematous indurations with possible features of necrosis in the center usuallydeveloping in a second time (6, 16). The exact reasons underlying these reactions are still debated. Similar manifestations have been reported with subcutaneous injections of interferon beta-l a, suggesting that the same mechanisms involving receptor sites, immunological and proinflammatory properties of both interferons are likely to be identified as potential triggers of these phenomena (17, 18). Indeed, recently, the case was reported of a 44-year-old diagnosed with MS who developed septal panniculitis following interferon beta-la treatment after an improperly administered intramuscular injection (19). Also, in literature the case is described of a 43-year-old woman affected by MS, who self-administered interferon beta-la subcutaneously 3 times a week. After 18 months of treatment, patient was diagnosed with mixed panniculitis secondary to therapy (20). Therefore, it is noteworthy that development of septal panniculitis may be the result of treatment with other systemic drugs, such as interferon beta-la, not only interferon beta-lb. Some authors also speculated on the fact that such reactions occur even more frequently in association with IFN-~-lb compared to IFN-~-la. Immunologicall- mediated necrotizing vasculitis and platelet-dependent thrombosis in the dermis may play a role. Multiple sclerosis per se or its immunomodulatory treatment seems to trigger clotting abnormalities, as shown by a consistent activation of plateles in these patients (21). Moreover, authors demonstrated the direct induction of local chemokine expression and the associated immune cell extravasation caused by interferon beta in human skin biopsy specimen (22). Histologically, perivascular dermatitis or panniculitis have been observed, with or without thrombosisofthe dermal vessels and leukocytoclastic vasculitis with perivascular fibrinoid material and nuclear dust in some cases. Lymphocytes are the main cell population (7, 15,23). The release of inflammatory cytokines from
Int. J. Immunopathol. Pharmacol.
subcutaneous adipose tissue even after minimal trauma, as also proved in our case by the positivity of TNF-(1 staining, mainly in, the infiltrated inflammatory cells in damaged tissues, may play a role in the inflammatory response (24). Also, the positivity for TNF-(1 correlated with an intense signal immunolocalization for I-CAM1, in accordance with previous studies, showing that TNF-(1 induces the upregulation of ICAM-l (25, 26). ICAM-l is an adhesion molecule expressed in leukocytes and endothelial cells, which is involved in recruitment of inflammatory cells, such as leukocytes and lymphocytes (27). Also, mast cells are implicated in leukocyte recruitment and tissue damage, indeed histamine is released through their degranulation. Moreover, immunohistochemical localization for chymase was performed to detect the presence of mast cells, but the section did not stain for this marker, thus confirming that allergic reaction is not involved in the pathogenesis of these lesions. On the contrary, positive staining was found by immunohistochemical analysis for CD4 and for CD8 alpha, which play a key role in cell-mediated immunity. In agreement with other studies, this work should enhance cognition that severe septal panniculitiscan also occurs as an adverse effect, although rarely, following subcutaneous injection with interferon beta-lb. Thus, we suggest that early recognition and correction of the injection technique may help to prevent severe complications. Moreover,authorsunderlinetherole ofintradermal injection as possible trigger of the adverse immune reaction, suggesting deep cutaneous penetration of the drug to avoid these reactions; in fact, the use of adequate training and/or automated injection devices has .been associated with an estimated 60% rate of reduction in the incidence of adverse cutaneous side effects (12,17,24,28). Certainly, discontinuation of the drug is followed by a rapid recrudescence of the disease, so education of MS patients regarding the correct administration of DMTs is mandatory in daily management of this invalidating condition (24, 28). At the same time, physicians should be familiar with these lesions for prompt diagnosis and treatment of both cutaneous and neurological diseases.
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