Anti-cytokine autoantibodies preceding onset of autoimmune polyendocrine syndrome type I features in early childhood.

PURPOSE: Almost all patients with autoimmune polyendocrine syndrome (APS)-I have high titer neutralizing autoantibodies to type I interferons (IFN), especially IFN-ω and IFN-α2, whatever their clinical features and onset-ages. About 90 % also have antibodies to interleukin (IL)-17A, IL-17F and/or IL-22; they correlate with the chronic mucocutaneous candidiasis (CMC) that affects ~90 % of patients. Our aim was to explore how early the manifestations and endocrine and cytokine autoantibodies appear in young APS-I patients. That may hold clues to very early events in the autoimmunization process in these patients. METHODS: Clinical investigations and autoantibody measurements in 13 APS-I patients sampled before age 7 years, and 3 pre-symptomatic siblings with AIRE-mutations in both alleles. RESULTS: Antibody titers were already high against IFN-α2 and IFN-ω at age 6 months in one sibling-8 months before onset of APS-I-and also against IL-22 at 7 months in another (still unaffected at age 5 years). In 12 of the 13 APS-I patients, antibody levels were high against IFN-ω and/or IL-22 when first tested, but only modestly positive against IFN-ω in one patient who had only hypo-parathyroidism. Endocrine organ-specific antibodies were present at age 6 months in one sibling, and as early as 36 and 48 months in two of the six informative subjects. CONCLUSION: This is the first study to collate the onset of clinical features, cytokine and endocrine autoantibodies in APS-I infants and siblings. The highly restricted early autoantibody responses and clinical features they show are not easily explained by mere loss of broad-specific self-tolerance inducing mechanisms, but hint at some more sharply focused early event(s) in autoimmunization.

Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a recessive disorder resulting from mutations in the autoimmune regulator (AIRE). The patients' autoantibodies recognize not only multiple organ-specific targets, but also many type I interferons (IFNs) and most T helper type 17 (Th17) cell-associated cytokines, whose biological actions they neutralize in vitro. These anti-cytokine autoantibodies are highly disease-specific: otherwise, they have been found only in patients with thymomas, tumours of thymic epithelial cells that fail to express AIRE. Moreover, autoantibodies against Th17 cell-associated cytokines correlate with chronic mucocutaneous candidiasis in both syndromes. Here, we demonstrate that the immunoglobulin (Ig)Gs but not the IgAs in APECED sera are responsible for neutralizing IFN-ω, IFN-α2a, interleukin (IL)-17A and IL-22. Their dominant subclasses proved to be IgG1 and, surprisingly, IgG4 without IgE, possibly implicating regulatory T cell responses and/or epithelia in their initiation in these AIRE-deficiency states. The epitopes on IL-22 and IFN-α2a appeared mainly conformational. We also found mainly IgG1 neutralizing autoantibodies to IL-17A in aged AIRE-deficient BALB/c mice - the first report of any target shared by these human and murine AIRE-deficiency states. We conclude that autoimmunization against cytokines in AIRE deficiency is not simply a mere side effect of chronic mucosal Candida infection, but appears to be related more closely to disease initiation.

Association of innate immune activation with latent Epstein-Barr virus in active MS lesions.

OBJECTIVE: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. METHODS: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. RESULTS: We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFNα in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFNα was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFNα production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFNα production in vitro. CONCLUSION: These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFNα production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.

Measuring autoantibodies against IL-17F and IL-22 in autoimmune polyendocrine syndrome type I by radioligand binding assay using fusion proteins.

Autoantibodies against interleukin (IL)-17A, IL-17F and IL-22 have recently been described in patients with autoimmune polyendocrine syndrome type I (APS I), and their presence is reported to be highly correlated with chronic mucocutaneous candidiasis (CMC). The aim of this study was to develop a robust high-throughput radioligand binding assays (RLBA) measuring IL-17F and IL-22 antibodies, to compare them with current enzyme-linked immunosorbent assays (ELISA) of IL-17F and IL-22 and, moreover, to correlate the presence of these antibodies with the presence of CMC. Interleukins are small molecules, which makes them difficult to express in vitro. To overcome this problem, they were fused as dimers, which proved to increase the efficiency of expression. A total of five RLBAs were developed based on IL-17F and IL-22 monomers and homo- or heterodimers. Analysing the presence of these autoantibodies in 25 Norwegian APS I patients revealed that the different RLBAs detected anti-IL-17F and anti-IL-22 with high specificity, using both homo- and heterodimers. The RLBAs based on dimer proteins are highly reproducible with low inter- and intravariation and have the advantages of high throughput and easy standardization compared to ELISA, thus proving excellent choices for the screening of IL-17F and IL-22 autoantibodies.

Aicardi-Goutières syndrome presenting with haematemesis in infancy.

UNLABELLED: Aicardi-Goutières syndrome is a genetic childhood encephalopathy characterized by basal ganglia calcification, chronic cerebrospinal lymphocytosis and elevated cerebrospinal fluid interferon-alpha, mimicking acquired congenital viral infections. As more is discovered about the pathogenesis of Aicardi-Goutières, it is becoming evident that a dysfunction of the immune system is likely to be responsible for the disease phenotype. We describe a previously healthy 2-month-old female infant who presented with haematemesis and seizures and was subsequently diagnosed with Aicardi-Goutières syndrome. To our knowledge, this is the first documented case of Aicardi-Goutières syndrome presenting with haematemesis. The gastrointestinal tract is an area of high cell loss, revealing early signs of systemic inflammation and we postulate that a systemic proinflammatory milieu occurs in Aicardi-Goutières syndrome. CONCLUSION: Aicardi-Goutières syndrome can present with haematemesis, adding to the growing evidence that the Aicardi-Goutières syndrome spectrum encompasses an immune-mediated multisystem involvement. Gastrointestinal inflammation should also be considered in these patients and treated appropriately.

Expanded TCR Vbeta subsets of CD8(+) T-cells in late-onset myasthenia gravis: novel parallels with thymoma patients.

Little is known about pathogenesis -- and especially about involvement of CD8(+) T-cells -- in late-onset myasthenia gravis (LOMG). Remarkably, outstanding CD8(+) TCRVbeta-subset expansions were found in 64% and 72% of recent onset LOMG or thymoma-associated MG (vs. 16% with early-onset MG (p<0.0002); 21% in older controls (p<0.001)). In LOMG, ~25% of the expanded cells initially showed a naïve CD62L(+hi)/CD45RA(+) recent thymic emigrant (RTE)-like phenotype. These expansions associated significantly with IgG antibodies against cytomegalovirus (p<0.036), IL-12 and/ or IFN-alpha2 (p<0.03). The CD8(+) TCRVbeta expansions were stable over 5years, but RTE markers declined.

Measurement of neutralising antibodies to type I interferons by gene expression assays specific for type 1 interferon-inducible 6-16 mRNA.

Human type I interferon products have been approved for the treatment of several diseases, though neutralising antibodies against them may develop and reduce therapeutic efficacy. Traditionally, potencies of human interferons (IFNs) and of neutralising antibodies (NAbs) against them are quantified by antiviral assays. These are being increasingly replaced by less cumbersome and faster bioassay methods. Since IFNs exert their biological effects by binding to receptors on target cells and stimulating the expression of IFN-inducible genes, measurement of transcribed mRNAs can form the basis of functional bioassays. In this study we have used two approaches, quantitative reverse transcription-polymerase chain reaction (qPCR) and branched DNA (bDNA), to develop efficient, sensitive and robust non-viral assays to quantify type I IFNs per se and NAbs in sera from patients treated with either IFNbeta or IFNalpha2a. We show the rapid (4h) induction of the type I IFN-inducible 6-16 mRNA in A549 lung carcinoma cells is sensitively and reproducibly concentration-dependent for both IFNbeta and IFNalpha2a stimulation, is quantifiable by either approach, and is readily adaptable for the detection and measurement of NAbs against type I IFNs. Quantitative neutralisation of IFN-stimulated 6-16 mRNA expression was achieved in both assays when sera from patients receiving IFNbeta or IFNalpha2a therapy known to contain NAbs against these IFNs were tested. Their rapid and potentially automatable performance strongly suggests these assays could be used in a clinical setting to monitor the development of neutralising antibodies in patients receiving IFN therapy.

Hypothetical review: thymic aberrations and type-I interferons; attempts to deduce autoimmunizing mechanisms from unexpected clues in monogenic and paraneoplastic syndromes.

In sporadic autoimmune disorders, dendritic cells are increasingly being incriminated as agents provocateurs. However, the mechanisms and any 'danger signals' that induce them to autoimmunize remain enigmatic. Here, we focus on unexpected clues from two prototypic/ highly informative autoimmune syndromes, acquired thymoma-associated myasthenia gravis and the monogenic autoimmune polyendocrine syndrome type-1 (APS1), caused by mutations in the AutoImmune Regulator (AIRE). Both involve the thymus, and in both we find early, persistent, highly prevalent and high-titre neutralizing autoantibodies against type-I interferons, regardless of the exact AIRE genotype or the characteristically variable clinical phenotype in APS1. Thus these key innate<-->adaptive immune intermediaries are now implicated in APS1 and paraneoplastic myasthenia as well as in systemic lupus erythematosus and other sporadic autoimmune disorders. The currently accepted notion that autoimmunization proceeds automatically (by 'default') does not explain how, when or where autoimmune responses are initiated against which targets in APS1, or whether exogenous or internal danger signals are involved, or predict whether the primary auto-immunogenic targets are AIRE-dependent. As the parallels between these syndromes must hold novel clues to these puzzles, they demand explanations. To unify these and other findings, we propose that autoimmunization occurs centrally in aberrant thymic environments rendered 'dangerous' by AIRE-deficiency (possibly by excess undegraded nucleic acids/dead cell debris). The ensuing autoreactivity focuses early on the locally abundant type I interferons and then on other peripheral tissue autoantigens that are still expressed despite the absence of AIRE. These ideas raise numerous questions that others may already have the materials to address.

AIRE variations in Addison's disease and autoimmune polyendocrine syndromes (APS): partial gene deletions contribute to APS I.

Autoimmune Addison's disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions of the AIRE gene covering at least exon 2 to exon 8. We conclude that polymorphisms in the AIRE gene are not associated with AAD and APS II. We further suggest that DNA analysis of the parents of patients found to be homozygous for mutations in AIRE, always should be performed.

Deficiency of the autoimmune regulator AIRE in thymomas is insufficient to elicit autoimmune polyendocrinopathy syndrome type 1 (APS-1).

Thymomas are thymic epithelial neoplasms, associated with a variety of autoimmune disorders (especially myasthenia gravis), that apparently result from aberrant intra-tumourous thymopoiesis and export of inefficiently tolerized T-cells to the periphery. The autoimmune regulator (AIRE) drives the expression of self-antigens in the thymic medulla and plays an essential role in 'central' tolerance in both humans and mice. However, while inactivating AIRE mutations result in the 'autoimmune polyendocrinopathy syndrome type 1' (APS-1), its major features are not well reproduced in AIRE-knock-out mice. Therefore, alternative human disease scenarios with concomitant AIRE deficiency may be valuable tools to test conclusions drawn from mouse models. Here we show, in a large series, that approximately 95% of thymoma patients are 'chimeric'; expression of AIRE and major AIRE-related autoantigens (eg insulin) were undetectable in their tumours but maintained in their remnant thymic tissue and lymph nodes. Notably, despite the AIRE-deficient thymopoiesis in thymomas, disorders and autoantibodies typical of APS-1 were distinctly uncommon in these patients. The one striking similarity was in the recently observed neutralizing anti-type I interferon (IFN) antibodies, which are found at diagnosis in 100% of patients with APS-1 and in approximately 60% of patients with thymomas, as we show here. We conclude that APS-1 type autoantigens must be protected from autoimmunity by mechanisms that do not extend to the muscle autoantigens so frequently targeted in thymoma patients but so rarely recognized in APS-1. Thus our findings argue strongly for a tolerogenic function of AIRE beyond its role in negative T-cell selection in human thymopoiesis, and/or for specific autoimmunization against muscle in thymomas.

Scenarios for autoimmunization of T and B cells in myasthenia gravis.

We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.

Autoantibodies to IL-12 in myasthenia gravis patients with thymoma; effects on the IFN-gamma responses of healthy CD4+ T cells.

In humans, interleukin-12 (IL-12) and interferon-alpha (IFN-alpha) normally favor IFN-gamma-producing "Th1" T cell responses. Myasthenia gravis (MG) patients with thymomas frequently have high-titer neutralizing autoantibodies against these cytokines, but not against IFN-gamma. Because they occasionally develop intractable (even fatal) infections, we have tested effects of their sera on the generation of IFN-gamma responses by healthy adult T cells to autologous lipopolysaccharide (LPS)-treated dendritic cells (DC). Anti-IL-12(+) sera consistently reduced IFN-gamma responses substantially, whether assessed by intracellular staining or ELISA. Therefore, thymoma patients with intractable infections might benefit from cautious IFN-gamma therapy. We discuss wider implications of the surprising rarity of clear clinical hazards-or benefits-of these autoantibodies.

Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis.

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

Establishment of new and replacement World Health Organization International Biological Standards for human interferon alpha and omega.

The complexity of the human interferon-alpha (IFN-alpha) family, with its multiple molecular forms and various biological activities, raises a number of scientific issues with regard to the biological standardisation of natural and recombinant IFN-alpha products. To address such issues and to achieve an appropriate biological standardisation of human interferon-alpha (IFN-alpha) preparations, the National Institute for Biological Standards and Control (NIBSC) of the United Kingdom (UK), in association with the Centre for Biologics Evaluation and Research (CBER) of the United States of America (USA), organised an international collaborative study, which was subsequently divided into two parts. Ninety-three participating laboratories from 29 countries worldwide participated in the first part of the study. They performed titrations on up to 15 different IFN-alpha preparations and one IFN-omega (omega) preparation in a variety of assays, including those based upon antiviral, antiproliferative, and other biological activities of IFN, and contributed raw data from these assays to NIBSC for analysis and calculation of relative activities. Analysis of data from this part of the study showed a greater than expected assay-dependent disparity between the relative activities of different IFN-alpha preparations. This disparity was found when only antiviral assays were considered and even when there were only small molecular dissimilarities between two otherwise closely related IFN-alpha preparations. The lack of assay independence and relative activity equivalence has indicated that a single biological potency standard for all IFN-alpha subtypes and mixtures would be inappropriate. Hence, individual, homologous standards, each with a separate unitage, were required for biological standardisation and potency determinations of individual IFN-alpha subtypes. At this stage, potency assignments to the IFN-alpha and -omega preparations included in the study were made as far as possible on the basis of comparison of antiviral activity with that of the 1st International Reference Preparation (IRP) for IFN, human leukocyte, 69/19. However, it was recognised that other standards had been used in assays to estimate potencies of widely available, current, therapeutic IFN-alpha products. Thus, to ensure the continuity of unitages already in use for IFN-alpha products, the second part of the study, which involved 12 members of the International Federation of Pharmaceutical Manufacturers Association (IFPMA), was carried out using for calibration of antiviral assays those IFN-alpha preparations that most closely matched manufacturers' products or that had been previously used for assay calibration by a manufacturer for a particular product. On the basis of data analysis from the second part of the study, potency assignments to the IFN-alpha preparations, as made in the first part of the study, were either left unchanged or changed to potency assignments that ensured as far as possible continuity with existing unitages. From among the IFN preparations evaluated, the following were recommended as the most suitable to continue or replace existing WHO international standards (IS) and have subsequently been formally established as WHO IS at the 51st meeting (October 1999) of the WHO ECBS: 83/514, 1st WHO IS for human IFN-alpha1 8000 international units (IU); 95/650, 2nd WHO IS for human IFN-alpha2a, 63,000 IU; 95/566, 2nd WHO IS for human IFN-alpha2b, 70,000 IU; 95/580, 1st WHO IS for human IFN-alpha2c, 40,000 IU; 95/572, 1st WHO IS for human IFN-alpha1/8, 27,000 IU; 94/786, 1st WHO IS for human IFN-alphaCon1, 100,000 IU; 94/784, 2nd WHO IS for human IFN-alpha (leukocyte), 11,000 IU; 95/574, 1st WHO IS for human IFN-alpha (leukocyte n3), 60,000 IU; 95/568, 2nd WHO IS for human IFN-alpha (lymphoblastoid n1), 38,000 IU; 94/754, 1st WHO IS for human IFN-omega, 20,000 IU. These WHO IS are available upon request to NIBSC.

Aicardi-Goutières syndrome displays genetic heterogeneity with one locus (AGS1) on chromosome 3p21.

We have studied 23 children from 13 families with a clinical diagnosis of Aicardi-Goutières syndrome. Affected individuals had developed an early-onset progressive encephalopathy that was characterized by a normal head circumference at birth, basal ganglia calcification, negative viral studies, and abnormalities of cerebrospinal fluid comprising either raised white cell counts and/or raised levels of interferon-alpha. By means of genomewide linkage analysis, a maximum-heterogeneity LOD score of 5.28 was reached at marker D3S3563, with alpha=.48, where alpha is the proportion of families showing linkage. Our data suggest the existence of locus heterogeneity in Aicardi-Goutières syndrome and highlight potential difficulties in the differentiation of this condition from pseudo-TORCH (toxoplasmosis, rubella, cytomegalovirus, and herpes simplex virus types 1 and 2) syndrome.

Effect of IFNbeta and anti-IFNbeta antibodies on NK cells in multiple sclerosis patients.

We analysed longitudinally the numbers of CD3-CD16+ (natural killer cells, NK) and CD3-CD57+ cells (a subset of NK) in 15 IFNbeta1b- and 12 IFNbeta1a-treated relapsing-remitting multiple sclerosis (RRMS) patients. IFNbeta1b (Betaferon)-treated RRMS patients showed a rapid and marked reduction in the number of both NK subsets which started 1 month after therapy initiation, and reached highest significance after 3 months (P=0.000); however, figures reverted to pre-treatment values following the appearance of anti-IFNbeta antibodies. In IFNbeta1a (Avonex)-treated RRMS patients, the decrease in both CD3-CD16+ and CD3-CD57+ cell number was slower but more persistent; anti-IFNbeta antibodies were only rarely detected in these patients, and at lower titers than in IFNbeta1b-treated ones. Our findings suggest that NK cells might be one of the major immunological targets of IFNbeta-based treatments.

Early events in TNFa-p55 receptor interations--experiments with TNF dimers.

The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule. To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer. For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E. coli and prepared in pure form. By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared. The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity. To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces. These dimers showed substantially reduced cytotoxicity on the L929 cell line. In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line.

Neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, interleukin-1alpha and interferon-alpha but not other cytokines in human immunoglobulin preparations.

Human immunoglobulin preparations are used therapeutically for various disorders. Such therapy is generally safe but adverse effects occasionally occur in recipients. It has been suggested that antibodies to cytokines present in clinical immunoglobulin products may contribute to undesirable effects in recipients. Therefore, we investigated intravenous and intramuscular immunoglobulin products for the presence of cytokine-specific neutralizing antibodies. Using validated bioassays, we detected neutralizing activity against human granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha2a (IFN-alpha2a) and interleukin-1alpha (IL-1alpha) in immunoglobulin products. We found no neutralization of granulocyte colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12, tumour necrosis factor-alpha, oncostatin M (OSM) and IFN-gamma. Most batches which neutralized IFN-alpha2a activity also neutralized other IFN-alpha subtypes, IFN-omega and IFN-beta. Most products (94%) neutralized the biological activity of GM-CSF. No correlation between batches and their ability to neutralize bioactivities of GM-CSF, IFN-alpha2a and IL-1alpha was found. This neutralizing activity could be traced to plasma pools used for manufacture of immunoglobulins. The neutralization was mediated by specific cytokine antibodies contained within immunoglobulin products as it was present in specific immunoglobulin G (IgG) fractions eluted from cytokine affinity chromatography columns. Specific binding of such IgG fractions to cytokines in immunoblots and in enzyme-linked immunosorbent assays (ELISAs) was observed. This contrasts with the broad non-specific recognition of cytokine proteins observed using unfractionated immunoglobulins in ELISAs. This is the first comprehensive study showing the presence of neutralizing antibodies against GM-CSF, IL-1alpha, or IFN-alpha2a in immunoglobulin products.

Human NK cells constitutively express membrane TNF-alpha (mTNFalpha) and present mTNFalpha-dependent cytotoxic activity.

We have evaluated the expression and the involvement of membrane-associated TNF-alpha (mTNF-alpha) in human NK cell-mediated cytotoxicity. Results from FCM analysis show that peripheral blood NK cells constitutively express mTNF-alpha. In contrast, mTNF-alpha expression is undetectable on resting T cells, B cells and monocytes. Western blotting analysis confirmed that freshly purified NK cells express the 17-kDa soluble form (sTNF-alpha) and the 26-kDa transmembrane form of TNF-alpha. Stimulation with IL-2, IL-15 and IL-18 up-regulates TNF-alpha mRNA, sTNF-alpha and mTNF-alpha expression in NK cells. The role of mTNF-alpha in the cytotoxic activity of resting NK cells has been evaluated in in vitro cytotoxic assays using freshly purified NK cells fixed with paraformaldehyde as effector cells (in order to avoid the participation of cytotoxic soluble mediators such as perforin, granzymes or sTNF-alpha) and the TNF-alpha-sensitive Fas ligand- and TRAIL-resistant cell line KYM-1-D4 as target cell. Results show that fixed NK cells kill the KYM-1-D4 cells and that neutralizing anti-TNF-alpha antibodies partly prevent this effect. In contrast to the other types of peripheral blood mononuclear cells NK cells from adult blood constitutively express functional mTNF-alpha in the absence of prior contact with target cells or activation. These data demonstrate a novel mechanism of cell-mediated cytotoxicity by non-acitvated human peripheral blood NK cells.