The avidity of PR3-ANCA in patients with granulomatosis with polyangiitis during follow-up.

The objective of this study is to investigate whether the avidity of proteinase-3-anti-neutrophil cytoplasmic antibody (PR3-ANCA) changes during follow-up in different subgroups of patients with granulomatosis with polyangiitis (GPA). We selected 10 patients with renal relapsing GPA, 10 patients with renal non-relapsing GPA and 10 patients with non-renal relapsing GPA. In all patients, an ANCA rise occurred during remission. The avidity was measured using a chaotropic approach at the time of an ANCA rise and at the time of a relapse in relapsing patients or time-matched during remission in non-relapsing patients. No difference was observed in the avidity at the ANCA rise between renal relapsing patients [26·2% (15·5-47·5)], renal patients without a relapse [39·6% (21·2-63·4)] and non-renal relapsing patients [34·2% (21·6-59·5)]. In renal relapsing patients, the avidity increased significantly from the moment of the ANCA rise to the relapse [difference 6·4% (0·0-17·1), P = 0·0273]. The avidity did not increase after an ANCA rise in renal non-relapsing patients [difference 3·5 (-6·0 to 10·1), P = 0·6250] or in non-renal relapsing patients [difference -3·1% (-8·0 to 5·0), P = 0·5703]. The avidity of PR3-ANCA increases after an ANCA rise during follow-up in renal relapsing patients, but not after an ANCA rise in renal patients who remain in remission or in non-renal relapsing patients.

Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension?

Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti-endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real-time cell electronic sensing (RT-CES™) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA-positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA-negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA-positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT-CES™ technology. IgG of AECA-positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA-negative PAH and SSc patients and healthy controls. AECA-positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECs in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody-dependent cell-mediated cytotoxicity.

Favorable long term effects of intensified immunosuppression combined with therapeutic plasma exchange in patients with early-onset progressive systemic sclerosis-related interstitial lung disease.

Objective: Systemic sclerosis (SSc) related mortality and morbidity remains high. Immunosuppressive therapy is considered most effective when immune activity and inflammation but not fibrosis still dominates the disease process. This study evaluated long-term intensified immunosuppression combined with therapeutic plasma exchange (TPE) in early-onset progressive SSc-related interstitial lung disease (ILD). Methods: The study cohort consisted of 161 SSc patients, with a median follow-up time of 8.9 years. The standardized mortality rate (SMR) and overall survival was calculated in patients with and without cardiopulmonary involvement. We used a standardized, pragmatic, non-randomized approach to treat 24 consecutive early progressive SSc-ILD patients with intensified immunosuppressive therapy, including plasma exchange. Outcome measurements were event-free survival (EFS), pulmonary function and safety profile. The outcome was compared with the analyzed data from the other SSc-ILD patients, who did not fulfill the inclusion criteria, and instead were treated with estimated optimal care (EOc). Results: The age-adjusted SMR of all 161 SSc patients was 3.0 (CI95%; 0.32-5.68). EFS at 10 years was 49.9% in the intensified treatment group and 43.3% in the EOc group (p = 0.106). Improvement of the percentage of predicted forced vital capacity (%pFVC) and percentage of predicted diffusing capacity for carbon monoxide (%pDLco) in the intensified treatment group was +10.1% respectively +3.6%, compared to a decrease of respectively 10.8% and 7% in the EOc (p < 0.001 resp. p = 0.019). Safety analysis showed 1 death (female patient, over 75 years of age), due to pneumosepsis, in the intensified treatment group. Conclusion: Intensified and long-lasting immunosuppression combined with TPE is safe in early severe systemic sclerosis and is associated with improved EFS and pulmonary function as compared to the outcome in the variable but EOc group. Our findings warrant larger studies for confirmation.

Sialoadhesin on macrophages: its identification as a lymphocyte adhesion molecule.

In this study we present evidence that the mouse and rat sialoadhesin (originally named sheep erythrocyte receptor) on macrophages can function as a lymphocyte adhesion molecule. Lymphocytes were shown to bind to the splenic marginal zone, and lymph node subcapsular sinus and medulla in a frozen section assay. Selective depletion experiments showed that binding was mediated by macrophages. Adhesion was blocked by preincubation of the sections with monoclonal antibodies against mouse or rat sialoadhesin. Binding was temperature dependent, divalent cation independent, and involved sialic acid residues on the lymphocyte, as it could be inhibited by prior neuraminidase treatment or addition of the ganglioside GD1a. Binding to sialoadhesin was confirmed using the purified receptor and was observed among T cells, T blasts, B cells, and B blasts. Isolated macrophages or dendritic cells showed little binding. Sialoadhesin provides the first example of a macrophage-restricted lymphocyte adhesion molecule.

Increased cytokines, chemokines and soluble adhesion molecules in exhaled breath condensate of asthmatic children.

BACKGROUND: Airway inflammation in asthma is characterized by the production of cytokines, chemokines and soluble adhesion molecules. The assessment of these inflammatory biomarkers in exhaled breath condensate (EBC) is hampered by low detection rates. However, the use of a glass condenser system combined with a sensitive analytical technique may increase the possibility to assess these biomarkers in EBC in a reliable way. OBJECTIVE: (1) To assess the detection rates of cytokines (IL-1alpha, -1beta, -2, -4, -5, -6, -10, -12p70, -13, -18, IFN-gamma, TNF-alpha), chemokines [MIP1alpha (CCL3), MIF, eotaxin (CCL11), RANTES (CCL5), IP10 (CXCL10), IL8 (CXCL8), MCP1] and soluble adhesion molecules [soluble intercellular adhesion molecule (sICAM), soluble vascular adhesion molecule (sVCAM)] in EBC of children with asthma and healthy control children; (2) To study the differences in the biomarker concentration between children with asthma and controls. METHODS: Sixty children were included: 31 asthmatics (71% atopic) and 29 controls. Exhaled breath condensate was collected using a glass condenser system. The inflammatory markers (IM) were analysed using multiplex immunoassay technology. RESULTS: Detection percentages of cytokines, chemokines and adhesion molecules ranged from 94% to 100%, except for eotaxin (CCL11) and RANTES (CCL5) (detection rates of 10% and 45% in healthy controls, respectively). The intra-subject variability of biomarkers in EBC in the group as a whole ranged from 5.2% to 35.0%. In asthmatics, the levels of cytokines (IL-2, -4, -5, -6, -13, IFN-gamma), chemokines (MIP1alpha [CCL3], MIF, RANTES [CCL5], IP10 [CXCL10], IL8 [CXCL8], MCP1) and adhesion molecules (sICAM, sVCAM) were significantly increased in comparison with controls (P<0.05). CONCLUSION: If collected with a glass condenser and analysed by multiplex immunoassay technology, cytokines, chemokines and soluble adhesion molecules can be reliably demonstrated in EBC of children. Most of these IM were elevated in EBC of asthmatics compared with controls.

A Dutch consensus statement on the diagnosis and treatment of ANCA-associated vasculitis.

INTRODUCTION: Despite the availability of several guidelines on the diagnosis and treatment of antineutrophil cytoplasmic antibody-associated vasculitis (AAV), clinical routine practice will only improve when an implementation strategy is in place to support clinical decision making and adequate implementation of guidelines. We describe here an initiative to establish national and multidisciplinary consensus on broad aspects of the diagnosis and treatment of AAV relevant to daily clinical practice in the Netherlands. METHODS: A multidisciplinary working group of physicians in the Netherlands with expertise on AAV addressed the broad spectrum of diagnosis, terminology, and immunosuppressive and non-immunosuppressive treatment, including an algorithm for AAV patients. Based on recommendations from (inter)national guidelines, national consensus was established using a Delphi-based method during a conference in conjunction with a nationally distributed online consensus survey. Cut-off for consensus was 70% (dis)agreement. RESULTS: Ninety-eight professionals were involved in the Delphi procedure to assess consensus on 50 statements regarding diagnosis, treatment, and organisation of care for AAV patients. Consensus was achieved for 37/50 statements (74%) in different domains of diagnosis and treatment of AAV including consensus on the treatment algorithm for AAV. CONCLUSION: We present a national, multidisciplinary consensus on a diagnostic strategy and treatment algorithm for AAV patients as part of the implementation of (inter)national guideline-derived recommendations in the Netherlands. Future studies will focus on evaluating local implementation of treatment protocols for AAV, and assessments of current and future clinical practice variation in the care for AAV patients in the Netherlands.

Anti-oxLDL antibody isotype levels, as potential markers for progressive atherosclerosis in APOE and APOECD40L mice.

In humans and animal models of atherosclerosis, antibodies against oxidized LDL have been associated with atherosclerotic lesion development. It has been suggested that IgM anti-oxLDL antibodies are anti-atherogenic, whereas IgG anti-oxLDL antibodies are pro-atherogenic. In this study, we examined the relation between IgM and IgG antibody levels and atherosclerosis severity in APOE(-/-)CD40L(-/-) mice, which are deficient for IgG and develop moderate advanced atherosclerosis, and compared results with mice developing severe (APOE(-/-)) or no atherosclerosis (C57Bl/6). Mice were followed in time for anti-oxLDL antibodies while on high-fat diet or normal chow. Anti-oxLDL antibody levels were determined by ELISA. Results revealed that 24-week-old APOE(-/-)CD40L(-/-) mice had enhanced IgM anti-oxLDL antibody levels when compared with wild-type mice, but similar levels to those of APOE(-/-) mice. As expected, IgG anti-oxLDL antibody levels were almost absent in APOE(-/-)CD40L(-/-) mice. The transition from early to advanced lesions in APOE(-/-) mice was reflected by elevated IgM anti-oxLDL antibody levels. IgM anti-oxLDL levels did not further increase during progression to more advanced lesions. No relation was found between IgG anti-oxLDL levels and atherosclerosis severity. In conclusion, the severity of advanced atherosclerosis in mice is not reflected by IgM and/or IgG anti-oxLDL antibody levels. Furthermore, less advanced atherosclerotic lesion development in APOE(-/-)CD40L(-/-) mice does not seem to be the result of higher levels of protective IgM anti-oxLDL antibodies. Therefore, our study does not support the idea that the previously observed inconsistency in the relation between anti-oxLDL and atherosclerosis severity is due to differences in antibody isotypes.

Progress towards non-invasive diagnosis and follow-up of celiac disease in children; a prospective multicentre study to the usefulness of plasma I-FABP.

This prospective study investigates whether measurement of plasma intestinal-fatty acid binding protein (I-FABP), a sensitive marker for small intestinal epithelial damage, improves non-invasive diagnosing of celiac disease (CD), and whether I-FABP levels are useful to evaluate mucosal healing in patients on a gluten-free diet (GFD). Ninety children with elevated tTG-IgA titres and HLA-DQ2/DQ8 positivity were included (study group). Duodenal biopsies were taken, except in those fulfilling the ESPGHAN criteria. Plasma I-FABP levels and tTG-IgA titres were assessed sequentially during six months of follow-up. Eighty children with normal tTG-IgA titres served as control group. In 61/90 (67.8%) of the children in the study group an increased I-FABP level was found; in all these children CD diagnosis was confirmed. Interestingly, in 14/30 (46.7%) children with slightly elevated tTG-IgA titres (<10x upper limit of normal), an increased I-FABP level was found. In all these children the diagnosis of CD was confirmed histologically. After gluten elimination for six weeks I-FABP levels had decreased towards levels in the control group. Measurement of plasma I-FABP, in addition to tTG-IgA, EMA-IgA and HLAtyping, enables non-invasive diagnosing of CD in a substantial number of children, and might therefore be of value in the diagnostic approach of CD.

From ANA to ENA: how to proceed?

Anti-nuclear antibodies (ANA), as detected by indirect immuno-fluorescence, are hallmarks of autoimmune connective tissue diseases. Identification of the specificity for extractable nuclear antigens (ENA) is warranted because this may further differentiate between the distinct types of autoimmune connective tissue diseases. In recent years several different ENA, as recognized by ANA, have been identified and the knowledge of the molecular structure has been expanded. Together with technical developments this has enabled the introduction of several new anti-ENA antibody detection systems. In this review we will discuss the main logistic aspects of anti-ENA antibody testing that have to be solved in order to come to a consensus in order to deal with new developments in this field. We conclude that: 1. a positive ANA test should, depending on the titre and pattern, be followed by an anti-ENA antibody assay, 2: to fully appreciate the value of the new anti-ENA antibody detection systems a large, multicenter clinical evaluation is required, and 3: proper interpretation of reported test results requires that the clinician is aware of the way anti-ENA antibodies are detected and reported.

Leukocyte CD40L deficiency affects the CD25(+) CD4 T cell population but does not affect atherosclerosis.

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.

Cellular binding mechanism on rat macrophages for sialylated glycoconjugates, inhibited by the monoclonal antibody ED3.

The mAb ED3 recognizes a subpopulation of rat macrophages, with a highly restricted tissue distribution. The tissue distribution as well as the in vitro expression of the ED3 antigen and of the sheep erythrocyte receptor (SER), binding unopsonized erythrocytes in the mouse, are very similar. This receptor has almost the same binding characteristics, although a different tissue distribution, as the sialic acid binding receptor (SAR), binding ganglioside-coated erythrocytes in the rat. In this study we summarize the available literature concerning these sialic acid binding receptors (SER and SAR). Furthermore we have identified ED3 as SER by inhibition studies of erythrocyte binding with mAb ED3, as well as by the newly developed equivalents ED16 and ED17. We also show that light trypsin treatment of alveolar macrophages, expressing SAR, results in SER-like activity. This obtained SER-like activity could not be blocked by the mAb ED3, indicating that SER and SAR are different receptors. It appears that rat macrophages can express two receptors for sialylated glycoconjugates, a high-affinity receptor SER, recognized by mAb ED3, and a low-affinity receptor SAR, not recognized by mAb ED3.

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18).

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzyme-histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.

Costimulatory molecules CD80 and CD86 in the rat; tissue distribution and expression by antigen-presenting cells.

The CD28-CD80/CD86 costimulatory pathway provides a critical signal for T cell activation. Only recently rat CD80 and CD86 have been cloned and monoclonal antibodies have been generated. In this study we examined the expression of these molecules in lymphoid tissue and on purified subsets of antigen-presenting cells (APC). The target tissue of cyclosporin A-induced autoimmunity, i.e. the skin and tongue, were also examined for expression of CD80 and CD86. Whereas CD80 was hardly detected in the lymphoid tissues, CD86 was clearly expressed by non-lymphoid cells in the thymus, as well as in the secondary lymphoid organs. With respect to lymphoid cells, only germinal center B cells exhibited clear CD86 expression. Phenotypic analysis by flow cytometry revealed that only dendritic cells, both of thymic and splenic origin, expressed the full array of stimulatory molecules required for the proper activation of naive T cells. On development of cyclosporin A-induced autoimmunity, non-professional APC, i.e. epithelial cells, started to express MHC class II, but not the costimulatory ligands CD80 and CD86. However, CD86 staining was observed in the target tissue and was associated with Langerhans cells as well as infiltrating leukocytes. Altogether, our results show that also in the rat strong stimulatory capacity for primary immune responses is associated with the expression of the costimulatory ligands CD80 and CD86. As concluded from the in situ expression CD86 may be the predominant costimulatory ligand early in immune responses.

Rat bone marrow and monocyte cultures: influence of culture time and lymphokines on the expression of macrophage differentiation antigens.

A set of seven monoclonal antibodies (moabs) has been shown to discriminate in situ between distinct subpopulations of macrophages in the rat. It is still controversial if this heterogeneity is caused by the existence of different lineages or by differentiation of a common precursor. In both cases, the differentiation process might be regulated by microenvironmental factors. The present study examines the expression of the macrophage markers recognized by the seven ED-moabs in bone marrow and monocyte cultures. Furthermore, the impact of culture time and stimulating factors on the antigen expression in these cultures was tested. The expression of the ED3 antigen is highly inducible in bone marrow cultures. Factors that might be responsible for the increased ED3 expression are investigated. This strong ED3 expression by bone marrow-derived macrophages is nearly absent by monocyte-derived macrophages. This implies that the ability to express ED3 is blocked before the macrophage precursor cells enter the circulation to become monocytes. The ED2 expression cannot be induced under the tested circumstances bone marrow macrophages in vivo do not express these antigens. In culture, these macrophages stain positive for these markers already after the first day of culturing. The other three antigens are expressed on all macrophages under all tested circumstances.

Surface exposure of phosphatidylserine during apoptosis of rat thymocytes precedes nuclear changes.

Cell surface exposure of phosphatidylserine (PS) during apoptosis serves recognition and removal of the dying cell by phagocytes. Loss of phospholipid asymmetry and PS exposure is investigated by immunocytochemistry and related to morphological changes. Loss of membrane asymmetry was determined on dexamethasone-treated rat thymocytes using the PS specific probe annexin V. Thymocytes incubated in the presence of dexamethasone were studied in time series during the execution of the apoptotic program. Thymocytes first start to expose PS at their cell surface. At this initial stage the barrier function of the plasma membrane remains intact. At a later stage the plasma membrane becomes leaky for compounds like propidium iodide and subsequently the cell disintegrates into apoptotic bodies. Microscopical evaluation of dexamethasone-treated thymocytes showed that the cells with an apoptotic morphology all bound annexin V. The cells with a normal viable morphology lacked annexin V binding except for those cells that started to shed small vesicles. These vesicles were positive for annexin V, indicating a local disturbance of the phospholipid asymmetry. The local exposure of PS is considered to be a very early event of apoptosis, preceding the full sequence of morphological changes at the ultrastructural level.

Comparison of detection techniques for cytokine reverse transcriptase polymerase chain reaction; digoxigenin-labeled polymerase chain reaction permits sensitive detection of cytokine mRNA in rat heart allografts.

The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32P-labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNgamma. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNgamma mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR.

X-irradiation of the thymus is not required for the induction of cyclosporine-induced autoimmunity.

The thymus-dependent model of cyclosporine-induced autoimmunity (CsA-AI) in the Lewis rat requires a lethal total body X-irradiation and rescue with syngeneic or autologous bone marrow and cyclosporine (CsA) administration for at least 4 weeks; two to three weeks after cessation of CsA, the animals develop a graft-versus-host-like disease. The obligatory role of the thymus in the etiology of CsA-AI has been established unequivocally, but the way in which disease is thymus dependent is a topic of debate. In the present study we demonstrate that the model of CsA-AI requires the presence of a thymus for at least 2 weeks after total body irradiation and CsA administration, but that X-irradiation of the thymus itself is not necessary to bring about disease. Transplantation of neonatal thymus shows in addition that in the absence of X-irradiation of the thymus, CsA therapy is required to generate autoreactive cells, but that disease occurs only if peripheral autoregulatory cells are eliminated by X-irradiation.

Effect of in vivo rapamycin treatment on de novo T-cell development in relation to induction of autoimmune-like immunopathology in the rat.

Cyclosporine (CsA) and FK506 are structurally unrelated immunosuppressants, but function in similar ways. FK506 and rapamycin (RAPA), on the other hand, have structural similarities, but act by different mechanisms to yield immunosuppression. Besides their immunosuppressive action, CsA and FK506 are known to interfere with T-cell development. CsA treatment after lethal X-irradiation and syngeneic bone marrow transplantation results in autoimmune disease, which is referred to as CsA-induced autoimmunity. In this study, we examined the effect of RAPA on T-cell development by flow cytometry and immunohistochemistry in female Lewis and Brown Norway rats. Irradiation and syngeneic bone marrow transplantation were performed before a 4-week course of RAPA administration to determine de novo T-cell development in relation to possible autoimmune phenomena. RAPA interfered with the maturation of thymocytes to the CD4+CD8+ DP stage, which resulted in a relative increase in TCRalphabeta(-) immature thymocytes, localized in a rim along the outer cortex. The thymus of RAPA-treated animals had a thinner cortex, leading to stronger thymic atrophy. In the periphery, only a few T cells were observed at the end of RAPA treatment. In the Lewis rat, a normal CD4/CD8 T-cell ratio and an increased Th1/Th2 ratio was observed within the T-cell population. Six weeks after cessation of RAPA therapy, the T-cell compartment was restored to normal, with respect to number and phenotype. In Brown Norway rats, however, T-cell areas were barely detectable at the end of RAPA treatment. The CD4/CD8 T-cell ratio was decreased as a result of a lower number of CD4 T cells; the Th1/Th2 ratio was increased but Th2 remained higher. Similar to Lewis rats, the situation was almost normalized 6 weeks after cessation of RAPA administration. However, Brown Norway rats, in contrast to Lewis rats, showed T-cell infiltration and concomitant induction of MHC class II in the submandibular salivary gland, as well as insulitis, in the pancreas. Possible relationships to Sjogren's disease and diabetes remain to be established.

The lesions of cyclosporine-induced autoimmune disease can be equally well elicited by CD4 or CD8 effector T cells.

Lethally irradiated Lewis rats reconstituted with syngeneic bone marrow and given cyclosporine for 4 weeks develop a graft-versus-host-like disease upon withdrawal of CsA. Autoreactive T cells inducing this thymus-dependent autoimmune disease, termed CsA-AI, are demonstrable by adoptive transfer, provided regulatory cells in recipient rats are eliminated. Earlier studies have not unequivocally defined the effector T cells responsible for development of CsA-AI. Some of these studies suggest that both CD4 and CD8 T cells are required, while other studies indicate disease transfer by CD4 or CD8 T cells only. To further clarify this issue, it was necessary to study putative effector T cells in a well-defined setting. Hence, adoptive transfer studies were designed wherein the effect of the T cells of interest could be studied without being influenced by T cells of unwanted origin. Accordingly, recipient rats were thymectomized prior to irradiation, lymph node cells (LNC) from diseased donor rats were depleted of CD4 or CD8 cells before adoptive transfer, and recipients were treated in vivo with CD4- or CD8-depleting mAb. The results showed that CsA-AI developed after adoptive transfer with LNC depleted of either CD4 or CD8 cells. Analysis of PBL and of histologic specimens confirmed the absence of the depleted subset. In both instances, the typical MHC class II expression on keratinocytes and the presence of ED1+ macrophages were identical to the lesions in the primary donors, where both CD4 and CD8 T cells were present. Analysis of the T cell Receptor beta-chain variable region repertoires revealed that their expression patterns in LNC of diseased donors or recipients was comparable to that in normal thymus or LNC--hence, there was no restricted BV repertoire. Taken in toto, our observations indicate that CsA-AI involves both CD4 and CD8 T cells, and that these subsets can generate identical macroscopic and microscopic signs of disease.

Effects of antibody reactivity to major histocompatibility complex (MHC) and non-MHC alloantigens on graft endothelial cells in heart allograft rejection.

BACKGROUND: To gain insight in the pathogenesis of vascular lesions in heart allograft rejection, we investigated effects of allosera reactive with major histocompatibility complex (MHC) or non-MHC alloantigens on graft endothelial cells (EC) in a rat transplantation model. METHODS: Anti-MHC and anti-non-MHC allosera were obtained from Brown Norway (RT.1(n)) recipients of a Lewis (RT.1(1)) or congenic LEW.1N (RT.1(n)) heart allograft respectively. Reactivity with endothelial alloantigens was studied in vitro using a series of three rat heart endothelial cell (RHEC) lines of Lewis origin. Phenotypic studies of MHC and non-MHC alloantigen expression, and adhesion molecule induction on EC were performed by immunostaining and fluorescence-activated cell sorting analysis. Complement-mediated cytotoxicity of allosera was studied using a 51Cr release assay. RESULTS: Both anti-MHC allosera and anti-non-MHC allosera showed reactivity with all three RHEC lines. EC stimulation with tumor necrosis factor-alpha and interferon-y resulted in increased reactivity of anti-MHC but not of anti-non-MHC allosera. Anti-MHC allosera showed complement-mediated cytotoxicity for EC, which was strongly increased when cytokine-stimulated EC were used. With anti-non-MHC allosera, only minor cytotoxicity was measured, irrespective of the activation of EC. Anti-MHC and anti-non-MHC allosera from the day of rejection (days 7-8 and days 29-35, respectively) had similar subclass profiles of allospecific IgG, except for allospecific IgM, which was only detected in anti-MHC allosera. Complement-mediated cytotoxicity of anti-MHC allosera from the day of rejection was effected mainly by IgM alloantibodies, whereas, in allosera taken 4 days after rejection, a predominance of cytotoxic alloantibodies of the IgG class was observed. No indications were found that either alloantibody reactivity alone or in combination with complement activation led to EC activation processes relevant to intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 induction. CONCLUSIONS: Our data show that, in heart allograft rejection, MHC but also non-MHC alloantigens on EC are target structures in the alloantibody response. Alloantibodies reactive with endothelial MHC, but not those reactive with non-MHC alloantigens, may significantly contribute to vasculopathy by complement-mediated cytotoxicity. Although no evidence was found that alloantibodies reactive with graft EC induce adhesion molecule expression, they may trigger other EC mechanisms relevant to graft vasculopathy.