Immunofluorescence studies on complement components in the hair follicles of normal scalp and of scalp affected by alopecia areata

The deposition of complement components (Clq, C4 C3, C5 and C9) and properdin in scalp hair follicles was examined by the immunofluorescence method in patients with alopecia areata and in normal subjects. C3, C5 and C9 were found deposited there. The complement deposition was most frequent in the case of C3 and less frequent in C9 and C5. There was no difference regarding deposition between normal subjects and patients with alopecia areata. No deposition of Clq, C4 or properdin was observed in the hair follicles of the scalp. These findings suggest a relationship between the hair cycle and the activity of C3 and its late complement components in the scalp.     

Mannan-binding lectin and ficolin deposition in skin lesions of pemphigus

Pemphigus is characterized by circulating autoantibodies directed against desmossomal antigens that, once bound to target antigens, induce disruption in the cell–cell adhesion of the epidermis and mucosal epithelium, leading to blister formation. Evidence has indicated a role for complement in the physiopathology of pemphigus, with complement deposition in intercellular spaces of skin and mucous membrane lesions. Mannan-binding lectin (MBL) and Ficolin-2 are recognition proteins of innate immunity, which by binding to specific molecular patterns on pathogens surfaces trigger the activation of complement, leading to phagocytosis and lyses of target cells and inflammation. In this study we report for the first time the deposition of MBL and ficolins in pemphigus lesions. Eight biopsies of skin lesions of pemphigus vulgaris were studied for in situ deposition of IgG and the complement components MBL, Ficolin 1, Ficolin-2, C1q, C3 and membrane attack complex C5b-9. All biopsies presented deposition of IgG and C3 in the intercellular spaces (ICS) of epidermis. MBL deposition was found in the ICS and basal membrane zone (BMZ) of all specimens, whereas C5b-9 showed deposition only in the ICS, with irregular distribution. Ficolin-2 were positive in 50% (4/8) of biopsies showing deposition in the BMZ. On the other hand, ficolin-1 and C1q were negative in all specimens. Our study suggest that MBL and to a lesser extend Ficolin-2 may bind to altered intercellular structures in the skin and lead to the activation of complement in situ, contributing to tissue damage in pemphigus.     

Vitronectin colocalizes with Ig deposits and C9 neoantigen in discoid lupus erythematosus and dermatitis herpetiformis, but not in bullous pemphigoid

C9 neoantigen immunoreactivity has been found to colocalize with C3 immunoreactivity at the dermal-epidermal junction zone (DEZ) in skin specimens from patients with bullous pemphigoid, lupus erythematosus and dermatitis herpetiformis. The present study was designed to elucidate whether the C9 neoantigen immunoreactivity represents deposition of membrane attack complexes or non-lytic SC5b-9 complexes. Skin specimens from 11 patients with pemphigoid, five patients with discoid lupus erythematosus and from nine patients with dermatitis herpetiformis were studied with immunofluorescence using both monoclonal and polyclonal antibodies against C9 neoantigen and against vitronectin (S-protein), an inhibitor to the membrane attack complex of complement. Specimens from the pemphigoid patients demonstrated C9 neoantigen reactivity along the DEZ without detectable colocalized vitronectin. This suggests deposition of membrane attack complexes in the pemphigoid lesions. Immunoreactivity of both C9 neoantigen and vitronectin was detected in the DEZ in specimens of discoid lupus erythematosus and in the tips of dermal papillae in specimens of dermatitis herpetiformis. The combined presence of C9 neoantigen- and vitronectin immunoreactivity may indicate deposition of C9 as part of the non-lytic SC5b-9 complex. The finding reported here of differential deposition of vitronectin and C9 in different diseases indicates that the presence of C9 neoantigen immunoreactivity in tissue per se does not represent the deposition of membrane attack complexes, but that it may also be C9 deposited as part of the nonlytic SC5b-9 complex.     

Erythema multiforme: demonstration of immune complexes in the sera and skin lesions

In twenty patients with erythema multiforme we investigated circulating immune complexes and their deposition in the skin lesions. C1q-binding activity was elevated in ten of twenty patients, and the platelet aggregation titre was high in three of twelve tested sera. Decreased levels of C3 were seen in two and of C4 in one out of eighteen patients. Direct immunofluorescence showed a deposition of C3, IgM or IgG in the blood vessel walls of the upper dermis in four of twelve patients. These findings may suggest that transient production of circulating immune complexes and their deposition play an important role in the pathogenesis of this disease.     

Complement activation by pulsed tunable dye laser in normal skin and hemangioma

Pulsed tunable dye laser (577 nm) (PTDL) therapy induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. To define whether this treatment activates complement in normal skin and senile hemangioma, we analyzed complement deposition in blood vessels by immunofluorescence. C3 fragments, C8, and C9 were detected with specific polyclonal antibodies. The membrane attack complex of complement (MAC) was demonstrated with a monoclonal antibody which reacts only with a neoantigen of MAC. Amplification of C3 deposition by the alternative pathway was determined on cryostat sections by indirect immunofluorescence with use of C4 deficient guinea pig (GP) serum. Normal skin and hemangiomas from three individuals were studied. In PTLD-irradiated normal skin, the main findings were as follows: 1) C3 fragments, C8, C9, and MAC were deposited in vessel walls; 2) these deposits were not due to denaturation of the proteins since they became apparent only 7 min after irradiation, contrary to immediate deposition of transferrin at the sites of erythrocyte coagulates; 3) the C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction which was specific since tissue necrosis itself did not lead to such amplification; 4) these reactions preceded the local accumulation of polymorphonuclear leucocytes. Tissue necrosis was more pronounced in the hemangiomas. The larger angiomatous vessels in the center of the necrosis did not fix complement significantly. By contrast, complement deposition in the vessels situated at the periphery was similar to that observed in normal skin with one exception: C8, C9, and MAC were detected in some blood vessels immediately after laser treatment, a finding consistent with assembly of the MAC occurring directly without the formation of a C5 convertase. These results indicate that complement is activated in PTDL-induced vascular necrosis, and might be responsible for the ensuing inflammatory response.     

CLq precipitin in the sera of patients with allergic vasculitis (Gougerot-Ruiter Syndrome).

The sera from two well-documented cases of allergic vasculitis were examined for the presence of C1q precipitins. Both sera contained material capable of precipitating C1q in agarose gel. The material from one of the sera was partially purified using ammonium sulfate precipitation. Sephadex G-200 filtration, and DEAE-cellulose chromatography. Attempts to identify the nature of C1q precipitin were unsuccessful, but it resembled the high-molecular-weight precipitins of sera from patients with systemic lupus erythematosus. In a lesion, deposition of fibrinogen, immunoglobulins, and complement were noted mainly in the vessel walls. No correlation between immunoglobulin and complement deposition in the skin and the presence of C1q precipitins in the blood could be established.     

Immunofluorescent studies of complement C3 in the hair follicles of normal scalp and of scalp affected by alopecia areata

The deposition of complement C3 in the hair follicles of scalps of 4 normal subjects and of the affected parts of 9 patients with alopecia areata was found by the direct immunofluorescent methods. No deposition of immunoglobulins (IgG, IgA, IgM, IgD and IgE) was found. In the normal scalps, complement C3 was found to be deposited in the hyaline membrane of the hair bulb and in the internal layer of connective tissue sheath at the lower about 2/3 transient portion of anagen follicles, and in the thickened hyaline membrane of the catagen follicles. The deposition of complement C3 was also found in the anagen and catagen-like hair follicles even in the patients with alopecia areata, which was similar to the finding in the normal scalps. From the fact that the deposition of complement C3 was found chiefly in the transient portion of the hair follicles of the normal scalps, and that the behavior of deposition of complement C3 reflected the morphological state of hair follicles in each stage of the hair cycle, even in alopecia areata, complement C3 may be intimately related to the hair cycle, even though its significance is as yet unknown.     

大疱性系统性红斑狼疮六例临床病理分析

目的 总结大疱性系统性红斑狼疮(BSLE)的临床特点、治疗及预后.方法 回顾性分析2009年5月至2015年9月诊治的6例BSLE患者临床资料.结果 男2例,女4例,平均发病年龄34岁.6例均表现为红斑或正常皮肤上出现张力性水疱、大疱,3例水疱、大疱排列呈环状.6例患者SLE疾病活动指数评分均大于4.皮损组织病理示表皮下水疱或裂隙;直接免疫荧光均表现为基底膜带线状IgG、IgM、IgA沉积,其中基底膜带线状C3沉积4例,线状C1q沉积2例.6例均予糖皮质激素、羟氯喹治疗;2例加用环磷酰胺.随访6例均发生程度不一的不良反应.结论 BSLE多见于中青年,皮损组织病理示表皮下水疱或裂隙,直接免疫荧光见基底膜带线状IgG、IgM、IgA或C3沉积.     

Complement C3 and immunoglobulin in inflammatory acne vulgaris

In patients with moderate to severe inflammatory acne, complement (C3) was detected by immunofluorescence in sixteen early inflammatory acne lesions but in only one of thirteen biopsies of non-inflamed skin from acne sites. C3 deposition occurred particularly in the walls of small dermal blood vessels and at the dermo-epidermal junction. IgM was identified in vessel walls in four of sixteen early lesions. In eight late inflammatory lesions C3 deposition was much less prominent and was present in vessel walls in only two. None of the late lesions showed vascular deposition of IgM. The observations indicate that complement activation occurs in inflammatory acne and it is suggested that this may play a pathogenic role in the inflammation.     

The inaccessibility of the outer membrane of adherent Treponema pallidum (Nichols strain) to anti-treponemal antibodies, a possible role of serum proteins.

Fresh and aged adherent T pallidum were used to study the accessibility of their outer membrane to antibodies by means of an indirect immunofluorescent technique. The integrity of the outer membrane was demonstrated by the non-reactivity with a monoclonal antibody directed against the axial filaments. Using the sera from patients with sero-positive primary and secondary syphilis no binding of IgG and IgM antibodies was observed. However, IgG and IgM antibody fractions isolated from the sera of patients with secondary syphilis, gave with the fresh fibroblast-adhering treponemes a mean of 14.5% IgG- and of 43.2% IgM positive treponemes. These means were 32.1% and 87.3% respectively for aged treponemes. Lower percentages were observed when fibronectin adhering treponemes were used. This demonstrates the inability of the outer membrane to bind antibodies in a majority of the fresh treponemes. This is partly lost on in vitro aging. Absence of IgG- and IgM fluorescence was also observed when sequential incubations with the antibody fractions and control sera were used. This was accompanied by the deposition of the third complement factor (C3) around the treponemes. Incubations of IgG- or IgM pre-coated adherent treponemes with heat-inactivated control sera or a C3 deficient serum did not result in the deposition of C3, and partially restored the detection of human antibodies. The most likely explanation for the absence of fluorescence is that antibodies become buried in an extra-cellular layer of serum proteins. The deposition of C3 from control sera alone most probably points to the classical pathway of complement activation and suggests that antibodies of rabbit origin constitute a part of the extracellular layer of treponemes.     

Vesicular pemphigoid--ultrastructural and immunoelectron microscopic study

We report a patient with vesicular pemphigoid who showed clinically numerous small vesicles and figurate erythema over the trunk and extremities. Although large blisters were absent, immunofluorescent study revealed IgG and C3 deposition at the basement membrane zone of the lesional skin. The patient responded well to the combined use of corticosteroid and dapsone. Immunoelectron microscopy showed IgG and C3 deposition at the undersurface membrane of the basal cells and the lamina lucida. Electron microscopy of the nonvesiculated early erythematous skin showed small vacuoles and lacunae along the uppermost portion of the dermis. Although the clinical features of this patient and the good therapeutic response to dapsone were atypical, the results of immunopathology and immunoelectron microscopy were consistent with typical pemphigoid.     

Adherence of Candida species to human epidermal corneocytes and buccal mucosal cells: correlation with cutaneous pathogenicity

Adherence of microorganisms to epidermal corneocytes may be a prerequisite for cutaneous colonization and infection. Six species of Candida were assayed in vitro for adherence to human epidermal corneocytes and buccal mucosal cells, and compared to previous studies of pathogenicity in a rodent model of cutaneous candidiasis. C. albicans and C. stellatoidea exhibited marked adherence to both epithelial cell types over time, and were cutaneous pathogens in the rodent model. The remaining species showed little or no adherence, and were nonpathogenic to skin. Adherence to corneocytes was not inhibited by ethylenediamine tetraacetic acid, mannan polysaccharide, or concanavalin A lectin. Fresh human serum, but not heat-inactivated serum, inhibited C. albicans adherence by 50%, and was associated with the deposition of complement components, C3 and factor B on blastospores. Adherence to epithelial corneocytes and mucosal cells is a property of pathogenic species of Candida, and may participate in cutaneous colonization and infection mechanisms. Adherence was time-dependent, and did not require divalent cations. Cell wall mannan may participate in the "adhesin" complex. Mannan activation of serum complement and deposition of C3 and factor B on blastospores may provide a protective action by inhibiting Candida adherence to corneocytes.     

Membrane attack complex of complement in Henoch-Schönlein purpura skin and nephritis

Summary The present study using direct immunofluorescence with monoclonal antibodies to C5b-9 complex-related antigens was undertaken to determine whether complement activation in Henoch-Schönlein purpura (HSP) causes assembly of the membrane attack complex of complement (MAC) in skin and nephritis lesions. The deposition of C5, C6, C7, C8, C9, and C5b-9 neoantigens was noted in the vascular walls of papillary dermis and/or subpapillary dermal plexus of the vessels in 11 out of 15 patients with HSP. Their presence in vessel walls indicates complement activation which leads to terminal complement activation. There were small deposits of S protein at the same sites in three of the 11 skin specimens. Thus, the majority of C5b-9 demonstrated in HSP skin was the cytolytically active C5b-9 complex, MAC. Granular deposits of C5b-9 related antigens without S protein were also found in the capillary walls and mesangium of the glomeruli of two out of four specimens from patients with HSP nephritis; in the other two S protein was colocalized with the deposition of C5b-9. The results of the present study indicate that complement activation leading to generation of MAC may possibly be involved in the pathogenesis of vascular injury in a significantly large number of skin lesions and of HSP nephritis.     

免疫酶组织化学法检测红斑狼疮皮损狼疮带研究

目的:探讨用免疫组化方法检测红斑狼疮皮损中的免疫球蛋白沉积对其诊断的价值。方法:用辣根过氧化物酶(HRP)标记抗体法分析研究387例红斑狼疮患者皮损中免疫球蛋白(IgG、IgA、IgM)及补体(C3、C1q)的沉积情况以及与临床诊断、组织病理学诊断的符合率。结果:临床诊断为红斑狼疮,且组织病理学诊断亦符合红斑狼疮的患者皮损中最常见、最易检出的狼疮带成分均为IgG、IgM,其次为C1q,C3与IgA不易检出;而临床诊断为红斑狼疮,但组织病理学诊断不符合的患者,皮损中几种免疫球蛋白和补体的检出率之间尚无明确差别。结论:免疫组化法检测狼疮带对红斑狼疮患者的临床和组织病理学诊断有重要参考价值,且此法简便易行,具有较好的可重复性。     

Dermo-epidermal blister formation by linear IgA dermatosis sera in normal human skin in organ culture

Fresh normal human skin samples were cultured at 37^°C for 48 hours with sera from 3 patients with linear IgA dermatosis and 2 patients with dermatitis herpetiformis (DH). Dermo-epidermal separation (DES) occurred in the skin samples cultured with the sera from linear IgA dermatosis patients, and linear IgA deposition was observed in the dermo-epidermal junction zone. However, no IgA deposition or DES was observed in the skin cultured with the sera of DH patients or normal individuals. The results were the same when sera of patients with linear IgA dermatosis were heated at 56^°C for 30 minutes before culturing with skin samples, but DES was prevented when a proteinase inhibitor, aprotinin (50%), or α₂-macroglobulin (10 mg/dl) was added to the cultures. Our results suggest that IgA deposition in the basement membrane and proteinases are involved in the formation of vesicles in patients with linear IgA dermatosis, but complements are not directly involved.     

Deposition of the membrane attack complex of complement in bullous pemphigoid

Bullous pemphigoid is associated with deposition of IgG and C3 at the dermal-epidermal junction. In order to see whether complement activation in bullous pemphigoid resulted in deposition of membrane attack complex (MAC) at the basement membrane zone, skin biopsies from patients with bullous pemphigoid were examined using a direct immunofluorescence technique. By employing a monoclonal antibody to a neoantigen of C9, the MAC was demonstrated in linear pattern at the basement membrane zone. These deposits were seen in both involved and uninvolved skin but the amount of MAC was greater in involved skin as judged by intensity of staining. Stippled deposits of MAC were also present in or around epidermal basal cells. The MAC could be generated in vitro by reaction of normal plasma with antibasement membrane antibody bound to sections of monkey esophagus. The IgG antibody activated complement and this complement activation proceeded all the way to the terminal step.     

Stratum corneum activation of complement through the antibody-independent alternative pathway

We provide evidence that stratum corneum (SC) activates complement through the alternative pathway to generate C5a anaphylatoxin. By immunofluorescence studies it was shown that in addition to circulating IgG autoantibody, there were anti-SC antibodies of IgM and IgA classes in the sera from normal individuals. However, all the titers were significantly lower than the level of C3 deposition between corneocytes. By contrast, no C1q deposition occurred. Immunoelectrophoretically the orthokeratotic SC homogenates were found to induce the conversion of C3 from native C3 to C3b in fresh human serum even when the classic pathway was blocked by Ca2+-chelation. Enzyme immunoassay showed that factor B split product, Bb, was generated by the SC homogenates in the Ca2+-chelated serum. Radioimmunoassay for C5a also demonstrated that the SC homogenates could generate C5a anaphylatoxin in serum to an extent similar to that in non-treated serum when restricted to the alternative pathway activation; neutrophil chemotactic activity was generated in Ca2+-chelated serum at levels comparable to that generated in non-treated fresh serum. We separated the SC samples into a cornified envelope and soluble and keratin fractions. The cornified envelope was more effective in activating complement. This activity resided in heat-stable and non-lipid substances of corneocytes. Our hypothesis is that when the SC comes in contact with serum, it activates complement mainly through the alternative pathway to induce chemotactic C5a anaphylatoxin. Hence, inflammation in normal individuals after a traumatic injury to the skin or rupture of acne comedones, or epidermal cysts and possibly the formation of subcorneal sterile pustules noted in several dermatoses are explainable through this mechanism.     

Urticaria and vasculitis: a continuum of histological and immunopathological changes

Histopathological criteria were used to classify twenty-four patients with chronic urticaria into three groups, which were then studied to establish whether circulating immune complexes (CICs), complement activation and deposition of immunoreactants are confined to patients with urticarial vasculitis. Group I (three patients) had classical urticarial vasculitis, and two of these patients showed hypocomplementaemia with evidence of C3 conversion and deposition of immunoreactants in lesional and uninvolved skin. Ten patients (group 2) with a dense perivascular mixed-cellular infiltrate had normal or raised complement levels and infrequent evidence of C3 conversion. Immunoreactants were detected only in their lesional skin. Eleven patients (group 3) had only a sparse perivascular infiltrate. In this group, complement was normal and immunofluorescence was essentially negative. Cryoglobulins were detected in group I patients only. Monoclonal rheumatoid factor and C1q binding were positive in all group I patients, half the group 2 patients and none of the group 3 patients. This study suggests that urticaria and urticarial vasculitis form a disease continuum, and identifies a group of patients with features intermediate between urticarial vasculitis and ordinary urticaria.     

Deposition of C3, C9 neoantigen and vitronectin (S-protein of complement) in lichen planus pemphigoides

We have compared the distribution of C3, C9 neoantigen (C9n) and vitronectin at the dermoepidermal junction in lichen planus pemphigoides with that in bullous pemphigoid. Eight out of 30 biopsies from patients with lichenoid lesions had linear C3 deposition at the basement membrane zone (BMZ); four of these patients had bullae and fulfilled the criteria for lichen planus pemphigoides. C9n immunoreactivity was detected as a linear or an intermittent linear/granular band at the BMZ only in these four patients, suggesting a role for the membrane attack complex of complement (MAC) in the pathogenesis of blister formation in lichen planus pemphigoides. Faint linear deposition of vitronectin, in addition to C9n, at the BMZ was seen in two of the four cases of lichen planus pemphigoides and three of six cases of bullous pemphigoid. This suggests that vitronectin may be deposited in association with C9n not only as part of the non-lytic SC5b-9 complex, but also as a regulatory step following the lytic action of MAC. A regulatory function for vitronectin in limiting tissue damage following activation of MAC is supported by our finding of a heavy deposition of vitronectin in association with C9n in a lichen planus pemphigoides patient in whom bulla formation had ceased.     

C4d immunohistochemical stain is a sensitive method to confirm immunoreactant deposition in formalin-fixed paraffin-embedded tissue in bullous pemphigoid

Background:  Bullous pemphigoid (BP) is characterized clinically by the onset of pruritic urticarial plaques, vesicles and bullae in a predominantly elderly population. While the diagnosis may be suspected on routine hematoxylin and eosin histology of formalin-fixed paraffin-embedded tissue, fresh-frozen tissue must be used to show the immunologic nature of the bullous process by direct immunofluorescence (DIF). The diagnosis is further confirmed and separated from epidermolysis bullosa acquisita (EBA) by subsequent serologic studies to detect antibodies directed against BP180 and BP230 antigens and characteristic antibody deposition on salt-split skin.
Methods:  Using a polyclonal complement fragment 4d (C4d) antibody, we stained formalin-fixed paraffin-embedded skin biopsy specimens from cases of BP and controls.
Results:  We showed characteristic linear basement membrane deposition of C4d in formalin-fixed paraffin-embedded tissue in seven of nine cases diagnosed as BP vs. EBA by DIF on fresh-frozen tissue. None of the four controls for which we had adequate tissue were positive.
Conclusion:  These results indicate that formalin-fixed paraffin-embedded tissue can be stained for the immunoreactant C4d to show characteristic immunoreactant deposition, potentially obviating the need for repeat biopsy for DIF and allowing clinicians to proceed to serologic confirmation of BP.