Eosinophil granule major basic protein 1 deposition in eosinophilic esophagitis correlates with symptoms independent of eosinophil counts.

In patients with eosinophilic esophagitis (EoE), symptoms often do not correlate with peak eosinophil counts (PEC) determined on histopathological examination of biopsy specimens. This may be because eosinophils degranulate during active disease and lose their morphological identity as intact cells and, therefore, are not enumerated on microscopic examination. Eosinophil granule proteins that are released into tissues with degranulation, including major basic protein 1 (eMBP1), likely contribute to disease pathogenesis and, therefore, may correlate with symptoms better than PEC. We sought to determine whether symptoms in patients with EoE more closely relate to eosinophil granule protein deposition than to eosinophil enumeration, especially in patients with fewer than 15 eosinophils per high power field (HPF). Esophageal biopsy specimens from 34 patients diagnosed with EoE were obtained for histopathological examination and for evaluation of eMBP1 staining by indirect immunofluorescence. PEC by histopathology were compared to extracellular eMBP1 grades by immunostaining. PEC and eMBP1 grades also were analyzed for their relationship to symptoms and clinical course. Biopsy specimens from 19 of the 34 patients had fewer than 15 PEC on histopathological examination, and the other 15 patients had 15 or greater PEC. Positive eMBP1 immunostaining was found in all symptomatic patients. EoE symptoms were related to eMBP1 immunostaining grades (p = 0.0001), but not PEC (P = 0.14). Eosinophil granule protein deposition, specifically eMBP1, is increased in esophageal biopsy specimens from symptomatic patients with EoE and may be a marker of disease activity, including patients with EoE who have 'resolved' disease.

Genome-wide linkage analysis of 160 North American families with celiac disease.

Celiac disease (CD) is a common autoimmune disease caused by exposure to the protein gliadin in wheat, and related prolamins in barley and rye. The prevalence of the disease in the US is 1:133. The aim of this study was to identify non-human leukocyte antigen (HLA) loci that predispose to CD. A genome-wide search of 405 microsatellite markers was performed on DNA samples from 160 families with a minimum of two cases of CD. Multipoint, parametric and non-parametric linkage (NPL) analyses were performed. Locations on chromosomes 1q, 3q, 6p, 6q, 7q, 9q and 10q showed linkage statistics (NPL scores or heterogeneity logarithm of the odds (HLOD) scores) of approximately 2.0 or larger. The greatest evidence for linkage outside of chromosome 6 was on 7q and 9q. An NPL score of 2.60 occurred at position 151.0 on 7q and a HLOD score of 2.47 occurred at position 144.8 on 9q under a recessive model. As expected, there was highly significant linkage to the HLA region on 6p, with NPL and HLOD scores exceeding 5.50. In conclusion, this genome-wide linkage analysis represents one of the largest such studies of CD. The most promising region is a putative locus on 7q, a region reported independently in previous genome-wide searches.

Neutrophil and eosinophil participation in atopic and vernal keratoconjunctivitis.

PURPOSE: A retrospective study was conducted at three centers to examine the participation of neutrophils and eosinophils in the inflammatory processes associated with atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). METHODS: Conjunctival specimens were obtained from four patients with AKC, six with VKC, and five normal subjects. Indirect immunofluorescent staining was used to localize neutrophil elastase (NE) and eosinophil granule major basic protein (MBP) in serial sections of all specimens. RESULTS: Specimens from both AKC and VKC patients revealed extracellular deposition of NE and MBP. Control specimens showed no or minimal extracellular NE, and no MBP. Statistical analysis demonstrated significantly greater extracellular MBP deposition in AKC specimens compared to controls (p = 0.009), and VKC specimens showed significantly greater staining for both extracellular MBP (p = 0.005) and NE (p = 0.006). CONCLUSIONS: These results suggest that neutrophils, in addition to eosinophils, play an active role in the pathogenesis of AKC and VKC as evidenced by the extracellular deposition of their specific granule proteins.

Simultaneous presence of mucous membrane pemphigoid and pemphigus vulgaris: molecular characterization of both autoantibodies.

There are several reports in the literature describing the coexistence of features of pemphigus vulgaris and pemphigoid in the same patient. We describe 15 patients with clinical, histological, and immunopathological features of mucous membrane (cicatricial) pemphigoid at the time of initial diagnosis. All 15 patients failed to respond clinically to conventional systemic agents over a mean period of 7.2 years. Hence, IVIg therapy was used. Prior to initiating IVIg therapy, features of mucous membrane pemphigoid and pemphigus vulgaris were demonstrated by various serological tests. Different assays were performed to identify molecular characteristics of these two autoantibodies. Twenty-five healthy normal individuals, 22 patients with mucous membrane pemphigoid, 17 patients with pemphigus vulgaris, and 12 patients with pemphigus foliaceus served as controls for comparison of serological studies. On indirect immunofluorescence, using monkey esophagous as substrate, sera of all 15 patients had demonstrable levels of anti-intercellular cement substance (ICS) or anti-keratinocyte cell surface antibody. Sera of 14 patients on salt split skin bound to the epidermal side of the split, which was consistent with mucous membrane pemphigoid. Sera of all 15 patients demonstrated binding to a 205-kDa protein (human B4 integrin) and a 130-kDa protein (desmoglein 3) on immunoblot. In a sample of sera from each of the 6 patients with mucous membrane pemphigoid and pemphigus vulgaris, the anti-ICS antibody was of the IgG4 subclass. The IgG4 subclass is a characteristic feature associated with pathogenic autoantibodies in pemphigus vulgaris. Hence, in such patients, a dual diagnosis should be considered and confirmed by various serological assays. It is possible that the presence of two pathogenic autoantibodies in these patients could have contributed to the lack of response to conventional immunosuppressive therapy.

Increased eosinophil infiltration and degranulation in colonic tissue from patients with collagenous colitis.

OBJECTIVE: Eosinophils infiltrate the colonic mucosa of patients with collagenous colitis (CC), although the pathogenetic implications are unknown, including whether these eosinophils are activated and degranulate in situ. We examined eosinophil infiltration and degranulation in the intestines of patients with CC by immunofluorescence for eosinophil granule major basic protein (MBP). METHODS: We used both conventional histology (hematoxylin and eosin) and indirect MBP immunofluorescence histochemistry on colon biopsy specimens from patients with CC (n = 21) and from healthy controls (n = 9). Scoring of histological features was performed on hematoxylin and eosin-stained sections on a 0 to 3 scale. Eosinophil infiltration and degranulation, as quantified by extracellular MBP staining, were scored in each specimen on a 0 to 4 scale. RESULTS: The inflammatory infiltrate of the lamina propria, the thickness of the collagen band, the numbers of intraepithelial lymphocytes, and degree of epithelial cell damage were all significantly increased in patients with CC as compared to controls (p < 0.0001). Scores for both eosinophil infiltration and degranulation were also significantly higher in the CC group compared to controls (p < 0.0001). The degree of infiltrating eosinophils by hematoxylin and eosin was correlated with eosinophil infiltration and degranulation by MBP immunostaining; however, no other correlations were found between eosinophil infiltration or degranulation by immunofluorescence and any of the histological parameters measured in the CC group. CONCLUSIONS: Eosinophil infiltration and degranulation are increased in the colonic mucosa of patients with CC compared to healthy controls. Eosinophils and their cytotoxic granule proteins could be involved in the pathogenesis of CC. Further studies will be necessary to elucidate the mechanisms of eosinophil activation in CC.

Allergic fungal sinusitis: an immunohistologic analysis.

BACKGROUND: Allergic fungal sinusitis is a noninvasive form of fungal sinusitis that has recently been delineated as a distinct clinicopathologic entity. It is increasingly recognized as a cause of chronic sinusitis, with the primary causative agents being members of the Dematiaceae fungus family. Although its immunopathogenesis has not been elucidated, the eosinophil is a prominent inflammatory cell on histologic examination. OBJECTIVE: We sought to characterize the involvement of eosinophils in sinus tissue and accompanying mucin from patients with allergic fungal sinusitis. As a comparison, neutrophil and mast cell involvement was also evaluated in the same group of patients. METHODS: Tissue specimens from 8 patients with allergic fungal sinusitis, along with 8 nasal polyp specimens from patients without allergic fungal sinusitis, were stained by using indirect immunofluorescence for eosinophil granule major basic protein (MBP). Neutrophil elastase and mast cell tryptase staining was also performed on the same allergic fungal sinusitis and nasal polyp tissues. RESULTS: MBP was diffusely localized within the mucin, showing intense staining at the periphery and variable staining of degenerated cell clusters throughout. Extracellular MBP in the mucin was strikingly greater than intact eosinophil staining. Diffuse extracellular neutrophil elastase was also present in the mucin. Mucinous areas showed no tryptase localization. Adjacent nonmucinous areas of respiratory mucosa showed predominantly cellular staining with eosinophil MBP, neutrophil elastase, and mast cell tryptase. MBP staining of nasal polyps showed a predominantly cellular pattern with focal areas of extracellular deposition. CONCLUSIONS: Given the known toxicities of eosinophil granule MBP and neutrophil elastase, their extracellular presence supports the contribution of these proteins in the pathogenesis of allergic fungal sinusitis and further indicates that eosinophil and neutrophil activation occurs in the disease.

Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils and monocytes.

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.

Pulmonary infiltrates after cytokine therapy for stem cell transplantation. Massive deposition of eosinophil major basic protein detected by immunohistochemistry.

Interleukin-2 (IL-2), a product of activated T-cells, is now being used in a number of protocols for cancer immunotherapy. In one stem cell transplantation protocol for breast cancer, IL-2 is used together with interferon-gamma (IFN-gamma) and cyclosporine to stimulate a graft-versus-tumor response and improve the likelihood of a prolonged remission. We present the case of a patient who developed peripheral eosinophilia, perihilar infiltrates, and hypoxemia after autologous stem cell transplantation and the use of recombinant IL-2 and IFN-gamma. Histologic analysis of transbronchial lung biopsies demonstrated a few eosinophils within the bronchial submucosa. Immunostaining using antibodies directed against eosinophil major basic protein (MBP), however, revealed massive extracellular deposition of this toxic granule protein throughout the lung parenchyma. IL-2 therapy is well known to induce a peripheral eosinophilia and to be associated with the capillary leak syndrome characterized by weight gain, edema, and oliguria. The findings noted in this case report suggest that the eosinophil activation that accompanies immunologic therapy with IL-2 can result in direct toxicity to the lung and a localized vascular leak syndrome. This syndrome should be considered in the differential diagnosis of pulmonary infiltrates that occur acutely after bone marrow transplantation with cytokine augmentation.

Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody.

The severe disease atypical measles occurred when individuals immunized with a poorly protective inactivated vaccine contracted measles, and was postulated to be due to a lack of fusion-inhibiting antibodies. Here, rhesus macaques immunized with formalin-inactivated measles vaccine developed transient neutralizing and fusion-inhibiting antibodies, but no cytotoxic T-cell response. Subsequent infection with measles virus caused an atypical rash and pneumonitis, accompanied by immune complex deposition and an increase in eosinophils. Fusion-inhibiting antibody appeared earlier in these monkeys than in non-immunized monkeys. These data indicate that atypical measles results from previous priming for a nonprotective type 2 CD4 T-cell response rather than from lack of functional antibody against the fusion protein.

Polymorphic eruption of pregnancy and herpes gestationis: comparison of granulated cell proteins in tissue and serum.

Polymorphic eruption of pregnancy (PEP) and herpes gestationis (HG) are pregnancy-related dermatoses of unknown aetiology with eosinophil infiltration which, at early stages, may show similar clinical and histopathological features. To determine the relative contributions of eosinophils, neutrophils and mast cells to the pathogenesis of PEP and HG through deposition of granule proteins, we studied tissue and serum from 15 patients with PEP and 10 with HG. Using indirect immunofluorescence with antibodies to human eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), neutrophil elastase and mast cell tryptase, we determined and compared cellular and extracellular staining patterns in lesional skin biopsy specimens and, using immunoassay, measured MBP, EDN, and ECP in patients' sera. Eosinophil infiltration and extracellular protein deposition of all three eosinophil granule proteins were present in both PEP and HG indicating a pathogenic role for eosinophils in both diseases. Staining for eosinophil granule proteins was especially prominent in urticarial lesions and around blisters in HG. EDN and ECP serum levels in PEP and ECP serum levels in HG were significantly increased compared with those in normal pregnant and normal nonpregnant serum. Neutrophils were more prominent in HG specimens than in PEP specimens; extracellular neutrophil elastase was minimally present and similar in both diseases. Mast cell numbers and extracellular tryptase deposition did not differ between the two diseases and did not differ from mast cell counts in skin of normal pregnant women. This study shows that eosinophil granule proteins are deposited extracellularly in tissue and are increased in serum in both PEP and HG. Moreover, eosinophil involvement in the two diseases is more consistent than neutrophil and mast cell involvement. Comparatively, tissue eosinophil infiltration and extracellular protein deposition is more extensive in HG than in PEP, suggesting that eosinophil involvement is greater in the pathogenesis of HG than PEP and similar to that found in bullous pemphigoid.

Severe subepidermal blistering disorder with features of bullous pemphigoid and herpes gestationis.

Herpes gestationis (HG) and bullous pemphigoid (BP) are blistering disorders with similar features, including urticarial lesions that progress to blisters and immunodeposition of C3 in a linear pattern at the basement membrane zone. Among their differences, HG is distinguished by its association with pregnancy, the puerperium, or hormonal perturbation. We describe the immunopathologic findings and clinical course in a multiparous woman with a severe blistering eruption. The patient was not pregnant. Malignancy evaluation was negative, and hormonal testing was normal. Histologic examination demonstrated a subepidermal bulla with eosinophils. Direct immunofluorescence showed C3 in a strong linear band at the dermal-epidermal junction. Indirect immunofluorescence demonstrated circulating IgG and IgG3 antibodies to basement membrane zone (epidermal component on salt-split skin), and complement-fixing IgG. Immunoprecipitation demonstrated antibodies to a 180-kd keratinocyte protein antigen. By clinical definition, this patient has BP. However, her disease presentation demonstrated features of both BP and HG.

Exaggerated arthropod-bite lesions in patients with chronic lymphocytic leukemia: a clinical, histopathologic, and immunopathologic study of eight patients.

BACKGROUND: Unusual papulovesicular lesions resembling arthropod bites have been described in patients with chronic lymphocytic leukemia (CLL). OBJECTIVE: Our purpose was to describe and characterize further the clinical, histopathologic, and immunopathologic features of these lesions. METHODS: Eight patients were identified retrospectively who had CLL and characteristic skin lesions. Clinical and histologic features were recorded. Skin biopsy specimens were analyzed immunohistochemically for eosinophil granule major basic protein, eosinophil-derived neurotoxin, neutrophil elastase, and mast cell tryptase. RESULTS: The clinical features, including the lesional distribution, suggested arthropod bites, although most patients could not recall having been bitten. Mixed T- and B-cell lymphoid cell infiltrates were present within lesions, along with prominent eosinophil infiltration and eosinophil granule protein deposition. CONCLUSION: Exuberant papulovesicular lesions develop in patients with CLL apparently as an exaggerated response to arthropod bites. Prominent eosinophil infiltration and degranulation within these lesions likely contribute to the severity of symptoms.

Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes.

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112+/-4 ng/10(6) cells, and ECP, 163+/-2 ng/10(6) cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.

Familial eosinophilic cellulitis, dysmorphic habitus, and mental retardation.

BACKGROUND: Eosinophilic cellulitis is a polymorphous, chronic disease characterized by eosinophil infiltration and granulomatous inflammation. OBJECTIVE: Our purpose was to describe the clinical, histologic, and immunohistologic findings in three family members who have had eosinophilic cellulitis since childhood associated with mental retardation and abnormal body habitus. METHODS: Family members were evaluated. Multiple skin biopsy specimens were obtained and examined after hematoxylin-and-eosin staining, by immunofluorescence and by electron microscopy. Blood specimens were analyzed by immunoassays for eosinophil granule proteins and eosinophil active cytokines. RESULTS: Three short-statured, mentally retarded family members with abnormal body habitus in at least two generations had recurrent eosinophilic cellulitis. Peripheral blood and bone marrow eosinophilia was present. Plasma eosinophil granule major basic protein and eosinophil-derived neurotoxin levels were elevated with normal plasma eosinophil cationic protein levels. Eosinophil survival in culture was increased by patients' plasma and was blocked with monoclonal interleukin-5 antibody. The level of plasma interleukin-5 was elevated. Lesional skin biopsy specimens showed massive staining for three eosinophil granule proteins. Electron microscopy showed eosinophil disruption. CONCLUSION: Eosinophilic cellulitis, mental retardation, and abnormal body habitus were likely inherited as a dominant syndrome in this family in which eosinophil involvement was striking.

Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9.

In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel layer. Matrigel transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.

Cutaneous injection of RANTES causes eosinophil recruitment: comparison of nonallergic and allergic human subjects.

RANTES, a member of the C-C chemokine family, is a potent chemoattractant for T lymphocytes and eosinophils, but not neutrophils. To determine the effect of RANTES on cell recruitment in vivo, we injected up to 4 microg of RANTES intradermally into both allergic and nonallergic subjects and obtained biopsies 30 min, 6 h, and 24 h later. A dose- and time-dependent recruitment of eosinophils, CD45RO+ cells, and CD3+ cells was observed, with no effect seen on polymononuclear, cutaneous lymphocyte Ag+, CD68+, or tryptase+ cells. Eosinophil recruitment occurred more rapidly in allergic subjects than in nonallergic subjects. No eosinophil infiltrate was observed in nonallergic biopsies at 30 min and 6 h, whereas significant eosinophil recruitment was observed in allergic subjects by 30 min, reaching near-maximum levels by 6 h. The peak responses at 24 h were similar in both groups (nonallergic, 110 +/- 24 eosinophils/mm2; allergic, 113 +/- 38 eosinophils/mm2). The two groups had comparable numbers of circulating eosinophils. Major basic protein staining demonstrated eosinophil degranulation in both allergic and nonallergic groups. RANTES injection resulted in activation of endothelial E-selectin expression at 24 h. Incubation of cultured HUVECs with RANTES had no effect on adhesion molecule expression, suggesting that the in vivo effect may have been indirect. Our studies demonstrate that RANTES is a potent chemoattractant for eosinophils, CD3+ cells, and CD45RO+ cells in human skin. The accelerated eosinophil recruitment in allergic subjects provides support for the hypothesis that eosinophils from these subjects are primed in vivo.

Dermal eosinophils in atopic dermatitis undergo cytolytic degeneration.

BACKGROUND: Immunofluorescent staining for eosinophil granule proteins in lesional skin of patients with atopic dermatitis shows extensive extracellular deposition throughout the upper dermis with relatively few intact eosinophils. OBJECTIVE: This study was carried out to determine whether eosinophil granule protein deposition in atopic dermatitis occurs by classical exocytosis, by piecemeal degranulation, or as a result of cytolysis. METHODS: Skin biopsy specimens from 10 patients with atopic dermatitis were examined by electron microscopy. RESULTS: The biopsy specimens showed varying degrees of dermal eosinophil granule major basic protein deposition by indirect immunofluorescence. Specimens from seven patients showed striking alterations of eosinophils by electron microscopy including intact eosinophils with granule alterations (reversal of core staining and/or core lucency) and with uropod processes. Biopsy specimens from six patients showed evidence of eosinophil degeneration with disruption of nuclear and/or plasma membranes. In four patients' specimens, membrane-bound eosinophil granules were present near degenerating eosinophils or were present in the absence of recognizable eosinophils. Evidence of classical exocytotic degranulation was not observed. Two of the specimens were also examined by immunoelectron microscopy for major basic protein localization. In these, major basic protein appeared to be lost from the granule core and distributed in the eosinophil cytoplasm as granules disintegrated and the cell disrupted. CONCLUSION: These findings support the hypothesis that eosinophils undergo cytolysis with release of granule contents and membrane-bound granules; this is likely the usual mechanism of eosinophil granule protein release in atopic dermatitis.