Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate.

BACKGROUND: The "International consensus statement on testing and reporting antineutrophil cytoplasmic antibodies (ANCA)" advocates screening by indirect immunofluorescence (IIF), but external quality assessment programmes often demonstrate different IIF patterns for a single serum. AIM: To determine whether the variation in IIF patterns can be attributed solely to errors in interpretation. METHODS: This study compared the IIF patterns produced by four sera (two with cytoplasmic or C-ANCA; one with perinuclear or P-ANCA with myeloperoxidase (MPO) specificity; and one P-ANCA without MPO specificity) that were tested in 11 different laboratories. The sera were examined according to individual laboratory protocols at dilutions of 1/10 to 1/40 using P1 (n = 4), P2 (n = 2), P3 (n = 2), or in house (n=3) neutrophil preparations and conjugates from manufacturers C1 (n = 3), C2 (n = 1), C3 (n = 2), C4 (n = 1), C5 (n = 2), and C6 (n = 2). The IIF patterns were noted in each laboratory, the testing repeated, and the fluorescent patterns photographed and subsequently discussed at a meeting of the Australian ANCA study group. RESULTS: All IIF patterns described in individual laboratories were confirmed on retesting and by the ANCA study group. Neutrophil substrates produced commercially or in house varied in their ability to demonstrate cytoplasmic granularity and interlobular accentuation, which distinguish between "C-ANCA" and "C-ANCA (atypical)". All commercial and in house neutrophil substrates demonstrated neutrophil nuclear extension of P-ANCA fluorescence, which correlates with MPO specificity. However, eight assays (eight of 43) from eight laboratories resulted in IIF patterns different from those usually seen. One of these produced a C-ANCA (atypical) rather than a C-ANCA pattern. The other seven resulted in at least some cytoplasmic fluorescence when the consensus pattern was P-ANCA with (n = 4) or without (n = 3) MPO specificity. These assays used three different commercial and one in house neutrophil substrate, and six different conjugates, with anti-IgG, anti-(Fab)'(2), anti-Ig (heavy and light chain), and anti-G, A, and M activity. Four of the seven assays tested on commercial substrates had used the manufacturer's conjugates. CONCLUSIONS: This study indicates that the variation in IIF patterns seen with ANCA positive sera tested in different laboratories does not necessarily result from errors in the interpretation of patterns and cannot be attributed solely to the use of a particular neutrophil substrate or conjugate, or to the use of substrate from one manufacturer and conjugate from another.

Cell free serum interleukin-2 receptor levels after heart transplantation.

The aim of this study was to predict episodes of rejection or infection in heart transplant recipients by monitoring serum interleukin-2 receptor (IL-2R) levels, rather than by endomyocardial biopsy. The shedding of IL-2R from activated lymphocytes results in increased serum interleukin-2 levels and is probably a result of immune activation occurring in both rejection and infection processes. As a group, heart transplant recipients who had no rejection of infection had significantly increased serum IL-2R levels compared with healthy controls. Patients who had moderate and severe rejection, and infection, had significantly increased serum IL-2R levels compared with levels in patients without rejection or infection. High serum IL-2R levels were seen mainly within the first 3 months after transplantation. Of 15 patients studied, nine had consistent increases in serum IL-2R levels with episodes of rejection or infection. Serum IL-2R levels did not correlate with serum cyclosporine levels. The monitoring of serum IL-2R levels was not a sensitive test of rejection because increases were not seen in all episodes of rejection; increases were seen, however, in all cases of infection. This test lacked specificity as serum IL-2R levels increased during episodes of both rejection and infection. In conclusion, the monitoring of serum IL-2R levels may be a useful way to routinely monitor or screen for possible rejection infection in heart transplant recipients, but this method should not replace endomyocardial biopsy for diagnosing rejection.

The role of alpha 1-antitrypsin deficiency in the pathogenesis of immune disorders.

The association between alpha 1-antitrypsin (alpha 1-AT) deficiency and a number of immune mediated diseases including rheumatoid arthritis, anterior uveitis, systemic lupus erythematosus, and asthma suggests that alpha 1-AT may be important not only as an anti-inflammatory protein but also as an immune regulator. That the relationship between decreased amounts of this inhibitor and these diseases is causal is suggested by both some of its physical properties and evidence indicating it is able to modulate immune function. alpha 1-Antitrypsin has a high plasma concentration, very broad range of inhibitory activity and is an acute phase reactant. Among other things, it is able to modulate lymphocyte proliferation and cytotoxicity, and monocyte and neutrophil function. Additionally, some of these changes are demonstrable in vivo in patients with severe alpha 1-antitrypsin deficiency. This paper reviews the important physicochemical characteristics of this protein, the association of its presence in decreased amounts with immune disorders, and finally the important mechanism that may underlie this disease association.

Familial occurrence of alpha 1-antitrypsin deficiency and Weber-Christian disease.

Severe panniculitis of the Weber-Christian type occurred in two brothers, both with marked alpha 1-antitrypsin (alpha 1-AT) deficiency and phenotype PiZZ. Studies of inflammatory and immunologic function were undertaken in these two patients as well as in a third brother with severe alpha 1-AT deficiency but without Weber-Christian disease. The findings of these investigations were suggestive of exaggerated immunologic and inflammatory function with enhanced lymphocyte responsiveness to phytohemagglutinin, enhanced activation of neutrophils and monocytes, and accelerated delayed hypersensitivity responses in all three subjects. This hyperreactivity may explain the apparent association of alpha 1-AT deficiency with Weber-Christian disease.

The effect of alpha 1 antitrypsin on the proliferative response of human peripheral blood lymphocytes.

In evaluating immune aberrations in patients with alpha 1 antitrypsin (alpha 1 AT) deficiency, we have previously shown that they exhibit enhanced lymphocyte responsiveness to PHA that is serum mediated. In this study, we demonstrate suppression of the PHA response by using purified alpha 1 AT, and also similar but less marked suppression of the Con A response. alpha 1 AT, however, has no effect whatsoever on PWM-induced proliferation. This effect is demonstrable provided alpha 1 AT is added within 4 hr of mitogen activation and is mediated by its action on adherent cells rather than on proliferating lymphocytes. Adherent cells still exhibit this effect if pulsed with alpha 1 AT, then thoroughly washed before their activation. This suggests that it may be inhibiting a membrane serine esterase already activated before the addition of PHA. Thus alpha 1 AT may modulate the activation of T cells through its effect on monocytes, leading to abnormalities in immunoregulation, and hence a predisposition to the development of a variety of immunologic disorders in alpha 1 AT-deficient subjects.

Eosinophilia VI, spontaneous synthesis of chemokinetics, chemotactic, complement receptor-inducing activities for eosinophils by bronchial t lymphocytes of asthmatic-bronchitic patients.

T lymphocytes were separated from the bronchial mucus of five patients with extrinsic asthma and chronic and mild bronchitis and who had numerous eosinophils in the bronchial mucus but no significant blood eosinophilia. The bronchial lymphocytes spontaneously, that is without treatment with antigens or mitogens, released into serum-free synthetic medium one or more substances in the molecular weight range of 30 000 to 60 000 daltons. These substances when tested on eosinophils of normal persons stimulated random movement (chemokinesis), attracted them (chemotaxis), enhanced their chemotactic response to activated complement, and increased the expression of C4 and C3b receptors. Chemokinetic and chemotactic activity for neutrophils was weak. No eosinophil-stimulating activity was found in cultures of bronchial lymphocytes treated with puromycin to inhibit synthesis. The blood lymphocytes did not spontaneously synthesize the substance(s). It is not known if the several eosinophil-stimulating activities are due to one or more substance(s), but the nature of the eosinophil responses, molecular weight and other features, indicate similarities with the 'eosinophil stimulation promoter-chemotactic' factor reported to be released from mouse or human lymphocytes treated with antigen. Eosinophil stimulation resulting in increased expression of specific receptors, and potential for non-specific adherence, by trapping or arresting randomly migrating cells, is believed to mediate accumulation of cells in an affected organ. Prolonged synthesis of such products as from activated lymphocytes in the lung, could account for much of the local eosinophilia.

Immunoregulation by alpha 1 antitrypsin.

alpha 1 antitrypsin (alpha 1 AT) deficiency is a common genetic disorder seen in about 10% of the population. It predisposes to the development of a large number of inflammatory and immunologic disorders including rheumatoid arthritis, systemic lupus erythematosus, juvenile chronic arthritis, anterior uveitis, ankylosing spondylitis, fibrosing alveolitis and emphysema. We have investigated immunologic function in subjects with severe alpha 1 AT deficiency and demonstrated serum mediated enhancement of lymphocyte response to PHA and increased zymosan activation of mononuclear cells and neutrophils as measured by their chemiluminescence. These patients also have accelerated delayed hypersensitivity responses and increased levels of factor B, C3 and C5 but normal levels of immunoglobulin and other complement components. Such abnormalities in immunoregulation demonstrate a tendency to hyperreactivity that may contribute to disease predisposition.

Eosinophilia. V. Delayed hypersensitivity, blood and bone marrow eosinophilia, induced in normal guinea-pigs by adoptive transfer of lymphocytes from syngeneic donors.

Dinitrochlorobenzene (DNCB) induced delayed hypersensitivity but no eosinophilia in guinea-pigs from two colonies. Citraconic anhydride (CA) induced delayed hypersensitivity and eosinophilia of the blood and bone marrow, and sites of skin tests were also infiltrated by eosinophils. In adoptive transfer of lymphocytes separated from peritoneal exudate cells of strain XIII-sensitized donors, lymphocytes from DNCB-sensitized guinea-pigs transferred antigen-specific delayed hypersensitivity; lymphocytes from CA-sensitized guinea-pigs transferred delayed hypersensitivity, and induced eosinophilia of the blood and bone marrow of the recipients. Treatment of the lymphocytes before transfer with antilymphocyte (thymocyte) globulin or puromycin suppressed the manifestations in the recipients; normal globulin did not. Active sensitization with DNCB induced formation of small amounts, and with CA larger amounts of anaphylactic antibody. Sera from the actively sensitized animals elicited no significant eosinophilia of blood or bone marrow in one group of recipients. Passive anaphylaxis elicited a transient eosinophilia of the blood, but not of the bone marrow. It is postulated that T-helper cells interact with B-lymphocyte precursors, particularly IgE B cells, to stimulate eosinopoeisis. This results in a reserve of mature eosinophils that may be released in any subsequent anaphylactic event.

The effect of acute and prolonged administration of prednisolone and ACTH on lymphocyte subpopulations.

The effect of acute and prolonged three week administration of prednisolone and ACTH on the numbers and function of T- and B-lymphocyte subpopulations in patients requiring corticosteroid therapy was studied. Prednisolone caused severe reduction in E-rosette-forming lymphocytes, phytohaemagglutinin response, EAC-rosette-forming lymphocytes and surface-membrane mu-positive B-lymphocytes maximal at 4--6 hr after administration with reversal sometimes to supernormal levels by 24 hr. Prolonged administration resulted in a similar pattern of response. Acute but not prolonged prednisolone administration caused a reduction in the percentage of E-rosette-forming lymphocytes maximal at 4 hr. ACTH caused moderate reduction in these parameters at 4 and 6 hr which remained low at 24 hr after prolonged administration.

Depressed lymphocyte function after bereavement.

During 1975 twenty-six bereaved spouses took part in a detailed prospective investigation of the effects of severe stress on the immune system. T and B cell numbers and function, and hormone concentrations were studied approximately 2 weeks after bereavement and 6 weeks thereafter. The response to phytohaemagglutinin was significantly depressed in the bereaved group on the second occasion, as was the response to concanavalin A at 6 weeks. There was no difference in T and B cell numbers, protein concentrations, the presence of autoantibodies and delayed hypersensitivity, and in cortisol, prolactin, growth hormone, and thyroid hormone assays between the bereaved group and the controls. This is the first time severe psychological stress has been shown to produce a measurable abnormality in immune function which is not obviously caused by hormonal changes.

T and B cell populations in blood and lymph node in lymphoproliferative disease.

Lymph node and peripheral blood lymphocytes were studied simultaneously for surface markers of T and B cells in 22 patients with lymphoproliferative diseases and 8 patients with non-neoplastic lymphadenopathy. This resulted in the classification of the malignancy from involved lymph nodes into 4 groups. Six patients had B cell lymphomata with normal or strong immunofluorescent staining for surface membrane immunoglobulin; 8 patients had B cell chronic lymphocytic leukaemia with pale staining for surface membrane immunoglobulin; 5 patients had T cell lymphomata and 3 patients were not definitely classifiable. In 6 out of 8 patients with B cell CLL, histopathology of lymph nodes showed infiltration with well differentiated lymphocytes and in all T cell lymphomata, the infiltrating cells were poorly differentiated. By the use of these markers, malignant lymphocytes were identified in the circulation in only 3 out of 6 patients with B cell lymphoma, in all patients with B cell CLL but in none of those with T cell lymphoma or unclassifiable lymphoma. Therefore a more conclusive characterization of the malignant lymphocyte in lymphoproliferative diseases must include an examination of involved lymph nodes.