Immunopathology of ocular sarcoidosis

Summary Sarcoidosis is a multisystem disease characterized by enhanced immune responses at sites of involvement. To elucidate the immunopathogenesis of ophthalmic lesions, cell infiltrates in biopsies from conjunctiva and other tissues involved (lungs, lymph nodes, skin) were studied in 26 patients with active sarcoidosis in order to define the surface phenotype and the distribution of cells in granulomatous lesions. Biopsy specimens were also stained for detection of immunoglobulins, complement and fibrinogen deposits. The data demonstrate a lymphocytes/macrophages interaction in the central core of granulomatous areas as the crucial event that initiates the maintains the state of inflammation: at all sites of disease activity is present a compartmentalization of T-cells expressing a helper-related phenotype which account for the great majority of infiltrating cells both in the early lesions (aggregate of macrophages surrounded by lymphocytic infiltrate) and in well-organized sarcoid granulomata. The presence of plasma cells and immunoglobulin deposits may represent an epiphenomenon in line with the helper infiltration, suggesting a local hyper-reactivity of the B-cells immune system. This study suggests some immunopathogenetic mechanisms leading to the formation and growth of conjunctival sarcoid granulomata.     

Functional analysis of cytotoxic cells in patients with acute nonlymphoblastic leukemia in complete remission

In the current study, we investigated the cytotoxic ability of peripheral blood mononuclear cells (PBMC) recovered from patients with acute nonlymphoblastic leukemia (ANLL) in complete remission (CR) against natural killer (NK)-sensitive, NK-resistant, autologous and allogeneic leukemic target cells taken at diagnosis. Our purpose was to define the role played by cytotoxic mechanisms in the control of leukemic cell growth in ANLL. Experiments were carried out at resting conditions and after in vitro activation with recombinant interleukin-2 (rIL-2) and anti-CD3 monoclonal antibody (moAb). At resting conditions, PBMC recovered from ANLL patients displayed a NK function that was not significantly different from controls (mean +/- standard error of the mean [SEM]: 21.9% +/- 3.9% versus control values of 27.5% +/- 2.9%; the P value was not significant [NS]), but they were unable to show cytotoxic activity against autologous and allogeneic leukemic cells. After in vitro boosting with rIL-2, PBMC were able to generate lymphokine activated killer (LAK) cells, as demonstrated by an increased killing of NK-resistant Daudi targets (16.3% +/- 2.7%). Although LAK activity was quantitatively lower than in control subjects (mean +/- SEM: 16.3% +/- 2.7% versus control values of 79.8% +/- 3.1%; P less than 0.001), it still exerted a cytotoxic effect against autologous and allogeneic leukemic cells. Similar results were obtained when anti-CD3 moAb was used as a stimulus in vitro. Our data suggest that nonspecific cytotoxic cells may be triggered to exert an in vitro cytotoxic effect on leukemic cells, which could possibly play a key role in vivo in the control of leukemic cell growth regulation.     

Generation of superoxide anion by alveolar macrophages in sarcoidosis: evidence for the activation of the oxygen metabolism in patients with high-intensity alveolitis.

We studied superoxide anion (O2) generation by alveolar macrophages (AM) isolated from bronchoalveolar lavages (BAL) of patients with sarcoidosis, and assayed immediately after the isolation or after maintenance in culture for 2 days. In assays of cells freshly isolated from BAL, AM of patients with active sarcoidosis with a high-intensity lymphocytic alveolitis produced more O2- in response to phorbol myristate acetate than AM of patients with inactive sarcoidosis. Also, after 2 days of cultivation sarcoid AM were heterogeneous in their capability to metabolize oxygen, although both AM of active and inactive sarcoid patients produced higher amounts of O2- than AM of healthy subjects. In vitro treatment with recombinant interferon-gamma (rIFN-gamma) caused an enhancement of the capability of AM of inactive sarcoid patients to produce O2- in response to PMA. AM of patients with active sarcoidosis did not respond to rIFN-gamma when they already produced O2- vigorously. However, they became sensitive to the activating effect of rIFN-gamma after the down-modulation of their capability to produce O2-, that occurred upon prolonged cultivation. Monocytes isolated from blood of sarcoid patients and assayed immediately or after different times of cultivation did not produce more O2- than control monocytes and monocyte-derived macrophages, thus indicating that the activation of AM in sarcoidosis is likely a local phenomenon. These studies strengthen the notion that T lymphocyte-macrophage interaction is a critical event in the pathogenesis of sarcoidosis and establish that the enhanced capability to metabolize oxygen to highly reactive intermediates by AM is one of the consequence of this interaction.     

Increased serum levels of soluble interleukin-2 receptor in patients with systemic lupus erythematosus and rheumatoid arthritis

In this study we investigated the serum levels of a released soluble form of the interleukin-2 receptor (sIL-2R) in 42 patients with rheumatoid arthritis and in 12 cases of systemic lupus erythematosus. Data were evaluated in relationship to the clinical phase and compared with those observed in normal controls (N=56) and in osteoarthritis (N = 7). Increased levels were observed in both rheumatoid arthritis (mean ± SE, 604±49 U/ml) and systemic lupus erythematosus (1438±481 U/ml). These values were significantly higher than in control (256±15 U/ml;P<0.001) and in osteoarthritis (298±33 U/ml;P<0.001) groups. In addition, the highest values were associated with the active phases of both rheumatoid arthritis (active vs inactive, 771±78 vs 451±39 U/ml;P<0.001) and systemic lupus erythematosus (active vs inactive, 2108±489 vs 499±75 U/ml;P<0.001). Our findings suggest that the detection of sIL-2R in rheumatoid arthritis and in systemic lupus erythematosus may represent a good marker of disease activity, which indirectly indicates the ongoing activation and/or proliferation of immunoreactive cells which are involved in the pathogenetic events of these autoimmune conditions.     

Rearrangement for the T-cell receptor gene and co-expression of immature T-cell markers and natural killer cell phenotype, in a patient with acute lymphoblastic leukaemia

We describe a patient with acute lymphoblastic leukaemia whose blasts co-expressed immature T-cell markers and nearly the entire phenotypic repertoire of NK cells. The T-cell nature of the proliferating blasts was proven by the demonstration of the rearrangement for the beta-chain of the T-cell antigen receptor. Although an abnormal phenotypic expression related to the neoplastic proliferation cannot be formally excluded, it is possible that the cells in this patient may represent the clonal expansion of a normal subpopulation of T-cell lineage NK-related cells frozen at an early stage of differentiation. These features provide arguments for discussing the controversial issue of the ontogeny of NK cells and their relationship to the T-cell lineage.     

Cell apoptosis and granulomatous lung diseases

Apoptosis, also known as activation-induced cell death or programmed cell death, is an active suicide mechanism that is involved in normal tissue turnover during embryogenesis and adult life. There are many examples of apoptosis in the immune system, including programmed cell death of T cells during negative intrathymic selection of the TCR repertoire and, in the postthymic phase, death of responsive T cells upon specific activation of the TCR/CD3 complex. Induction of apoptosis assures rapid disappearance of the immune response upon antigenic clearance, avoiding the metabolic costs involved in sustaining a large number of effector cells. The knowledge that failure of immune cells to die is the cause of a number of immune-mediated disorders has opened intriguing new avenues of exploration into the pathogenetic events leading to the accumulation of immunoinflammatory cells at sites of ongoing inflammation in granulomatous disorders, including granulomas initiated by infectious agents, such as Mycobacterium tuberculosis, or in sarcoidosis. In this paper we review recent results obtained in experimental animal models and patients with immune granuloma suggesting that the positive induction by ligands binding to membrane receptors or the induction or loss of intracellular suppressor signals regulates immunoregulatory mechanisms that drive the progressive development of the granulomatous structure. The great advances in understanding how mechanisms for the activation or downregulation of apoptosis have a pathogenetic role in the outcome of granulomatous disorders are also briefly considered.     

Expression and function of KIR and natural cytotoxicity receptors in NK-type lymphoproliferative diseases of granular lymphocytes

Using monoclonal antibodies (mAbs) specific for different natural killer (NK) receptors, we studied the lymphocyte population from 18 patients with NK-type lymphoproliferative disease of granular lymphocytes (LDGL). The analysis of both resting and cultured NK cell populations demonstrated that these patients are frequently characterized by NK cells displaying a homogeneous staining with given anti-killer Ig-like receptor (anti-KIR) mAb (11 of 18 patients). In most patients NK cells were characterized by the CD94/NKG2A+ phenotype, whereas only a minor fraction of the cases expressed CD94/NKG2C. In 7 of these patients we could also assess the function of the various NK receptors. Remarkably those KIR molecules that, in each patient, homogeneously marked the NK cell expansion were found to display an activating function as determined by cross-linking with specific anti-KIR mAb. The KIR genotype analysis performed in 13 of 18 cases revealed that in NK-type LDGL certain activating KIRs, as well as certain infrequent KIR genotypes, were detected with higher frequencies as compared to previously analyzed healthy donors. Moreover, most KIR genotypes included multiple genes coding for activating KIRs. The analysis of non-HLA-specific triggering receptors indicated that the natural cytotoxicity receptors (NKp46, NKp30) were expressed at significantly low levels in freshly drawn NK cells from most patients analyzed. However, in most instances the expression of NKp46 and NKp30 could be up-regulated on culture in interleukin 2. Our data indicate that in NK-LDGL the expanded subset is frequently characterized by the expression of a given activating KIR, suggesting a direct role for these molecules in the pathogenetic mechanisms of this disorder.     

JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4⁺IL-17A-F⁺ cells, as well as of T CD4⁺ cells expressing IL-23Rp19 (T CD4⁺ IL-23R⁺), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 Teff cells in SF of clinically active joints of PsA patients.