Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis.

Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex-mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.     

Mechanism of immune complex-mediated damage: induction of interleukin 1 by immune complexes and synergy with interferon-gamma and tumor necrosis factor-alpha

The ability of immune complexes derived from patients with type II mixed essential cryoglobulinemia to trigger the production of interleukin 1 (IL 1) was investigated. Immune complexes containing either IgM/IgG or IgA/IgG aggregates were shown to induce IL 1 production in human peripheral blood monocyte-enriched populations. Interferon-gamma alone did not induce detectable IL 1, but increased the IL 1 production induced by the immune complexes. Tumor necrosis factor-alpha, which is a potent inducer of IL 1, also enhanced IL 1 production following stimulation with suboptimal doses of the immune complex. The findings suggest that immune complexes may induce inflammation partly due to their capacity to induce the synthesis of IL 1.     

The inhibitory receptor FcgammaRII reduces joint inflammation and destruction in experimental immune complex-mediated arthritides not only by inhibition of FcgammaRI/III but also by efficient clearance and endocytosis of immune complexes

Studies of FcgammaRII-/- mice identified the inhibitory function of this receptor in joint inflammation and cartilage destruction induced with immune complexes (ICs). To extend our insight in the role of FcgammaRII in arthritis, we explored the role of FcgammaRII in the absence of activating receptors I and III using FcgammaRI/III-/- as well as FcgammaRI/II/III-/- mice. When antigen-induced arthritis (AIA) was elicited, which is a mixture of T cell and IC-driven inflammation, arthritis was almost absent at day 7 in FcgammaRI/III-/- mice. Remarkably, in FcgammaRI/II/III-/- mice, this model induced a tremendously increased arthritis as compared to wild-type controls. This implies that FcgammaRII regulates joint inflammation also in the absence of activating FcgammaRI and III. To confirm the IC specificity of this finding, similar studies were done with ICs or zymosan as arthritogenic stimuli. Strongly elevated inflammation was found in FcgammaRI/II/III-/- mice with IC but not with zymosan. Clearance studies identified accumulation of IgG in the knee joint in the absence of FcgammaRII. Moreover, macrophages expressing only FcgammaRII showed prominent endocytosis of preformed soluble ICs not different from controls. In total absence of FcgammaR (FcgammaRI/II/III-/-), macrophages completely failed to endocytose ICs. Although joint inflammation was much higher in AIA arthritic knee joints of FcgammaRI/II/III-/- and the inflammatory cells still expressed an inflammatory phenotype, severe cartilage destruction (MMP-mediated neoepitopes in the matrix and chondrocyte death) was completely prevented in contrast to the marked destruction which was observed in the wild-type. Our study indicates that FcgammaRII reduces joint inflammation in the absence of activating FcgammaR by promoting endocytosis and clearance of ICs from the joint. Infiltrating cells, which fail to express activating FcgammaR although they still become stimulated are no longer capable of inducing severe cartilage destruction.     

T lymphocyte interferon-gamma production induced by Plasmodium falciparum antigen is high in recently infected non-immune and low in immune subjects.

Interferon (IFN) alpha and gamma were measured by radio-immunoassays in supernatants from cultures of peripheral blood mononuclear cells (PBMC) or purified T cell subsets incubated with either Plasmodium falciparum schizont-enriched malaria antigen (mAg), uninfected red blood cells (RBC) or pokeweed mitogen (PWM). Cell donors were 24 clinically immune, healthy African adult native residents of a P. falciparum-endemic region, Haut-Ogooué, Gabon, and seven non-immune, European temporary residents with a history of a single to a few malaria infections during the previous 1 to 9 months. When PBMC were cultured in medium alone or with RBC antigen no or low titres of IFN-gamma were detected. PBMC proliferation and IFN-gamma production observed in the presence of mAg were dose dependent and significantly correlated. When cultured with mAg, PBMC from non-immune Europeans produced significantly higher levels of IFN-gamma than did PBMC from clinically immune Africans. No such difference was found when PBMC were cultured with PWM. The mAg-induced IFN-gamma production was due mainly to CD4+ T cells and was not enhanced by CD8+ T cell depletion. No IFN-alpha was detected in culture supernatants. Thus, P. falciparum antigens are able to induce in vitro production of IFN-gamma by CD4+ T cells; however, in this sample, individuals considered to be clinically resistant to malaria were low producers of IFN-gamma.     

Cell-mediated immune response during experimental arthritis induced in rats with streptococcal cell walls.

Chronic, remittent, erosive arthritis was produced in rats by a single intraperitoneal injection of an aqueous suspension of cell wall fragments isolated from group A streptococci. Arthritis could be induced in rats which had been immunologically compromised by neonatal thymectomy. Delayed hypersensitivity to cell wall peptidoglycan could not be elicited in these rats, although progressive joint disease was obvious by clinical and radiological measurements. A delayed skin test was elicited with peptidoglycan in non-thymectomized rats at 6 to 14 days after injection of low doses of cell wall fragments. Between 2 to 4 weeks after cell wall injection the skin test could not be elicited and these rats could not be sensitized again with peptidoglycan. After a high dose of cell wall the skin test could not be elicited at any time. These non-thymectomized rats which had been injected with cell walls remained hyporesponsive to peptidoglycan for at least 3 months. Lymphocytes from non-thymectomized cell wall-injected rats also showed a non-specific depression of lymphocyte response to phytohaemagglutinin in vitro, but this function was recovered between 2 to 4 weeks after cell wall injection. We conclude that cell-mediated immunity against bacterial cell wall antigens is not a pathogenetic factor in this experimental model of arthritis.     

Plasma antiviral activity and interferon-gamma production by superantigen-stimulated lymphocytes during normal human pregnancy

PROBLEM: Plasma interferon (IFN)-beta levels, lymphocyte responsiveness, and evaluation of the relationship between circulating antiviral activity (AA) and IFN-gamma production were studied in pregnant women and nonpregnant age-matched controls with the objective of elucidating the downregulation of IFN-gamma production in successful pregnancy. METHOD OF STUDY: In plasma and supernatants of peripheral blood mononuclear cell (PBMC) cultures, stimulated with staphylococcal enterotoxin A (SEA) superantigen, from 43 pregnant women with a history of normal pregnancy and 30 healthy nonpregnant age-matched controls, levels of AA were measured in a micromethod by inhibition of the cytopathic effect (CPE) caused by vesicular stomatitis virus (VSV) in the human amnionic cell line (WISH). RESULTS: Significantly higher plasma AA (60% was IFN-gamma and residual activity was acid-labile IFN-like) was present in pregnant women than controls. On the other hand, SEA-activated PBMCs from pregnant women produced significantly lower IFN-gamma levels than those of nonpregnant women. Furthermore, maternal plasma AA levels correlated negatively with IFN-gamma production by SEA-stimulated PBMCs. CONCLUSION: The hypothesis that successful pregnancy requires downregulation of IFN-gamma is only partially sustained, suggesting that the immunology of pregnancy is more complex and that murine and human pregnancy have different cytokine profiles.     

Regulatory immune responses induced by IL-1 receptor antagonist in rheumatoid arthritis

Anakinra, a human recombinant IL-1 receptor antagonist, is approved for the treatment of RA. In this study, 12 patients received the placebo plus MTX treatment, 38 patients received Anakinra combined with MTX treatment. Compared with the placebo plus MTX group, serum levels of IL-17, IFN-γ, IL-21 and IL-1β significantly decreased, the percentages of Th17 cells and Th1 cells were lower and the percentage of Treg cells was higher after receiving Anakinra combined with MTX treatment. The observed regulatory immune responses collectively correlated with clinical improvement in treated patients. A substantial response, ACR 20 at 24 w were consistent with those at 12 w, 16 w and 20 w, and was accompanied by a marked improvement in RA related laboratory parameters. The study reveals that the combination of Anakinra and MTX is safe and well tolerated, which induces regulatory immune responses and significantly provides greater clinical benefit than the placebo plus MTX group.     

Role of T cells in granuloma formation induced by Rhodococcus aurantiacus is independent of their interferon-gamma production

Intravenous injection of Rhodococcus aurantiacus into mice causes granulomatous inflammation dependent on endogenous interferon-gamma (IFN-gamma). This study investigated the mechanism of granuloma formation with an adoptive transfer system in IFN-gamma knockout (IFN-gamma(-/-)) mice. IFN-gamma(-/-) mice infected with R. aurantiacus did not develop granulomas, and high titres of endogenous interleukin-10 (IL-10) were detected in spleen extracts at 2 weeks after infection. The adoptive transfer of splenocytes from infected wild-type (IFN-gamma(+/+)) mice did not restore granuloma formation, although this treatment diminished IL-10 production in IFN-gamma(-/-) mice. Adoptive transfer of splenocytes from infected IFN-gamma(-/-) mice into infected IFN-gamma(+/+) reduced granuloma formation. These results suggest that splenocytes of IFN-gamma(-/-) mice suppress granuloma formation. On the other hand, although IFN-gamma production induced by R. aurantiacus infection was detected in nude mice, which are deficient in T cells, granuloma formation was not induced in them. However, adoptive transfer of immune splenocytes from either IFN-gamma(+/+) mice or IFN-gamma(-/-) mice could induce granuloma formation. This means that splenocytes of IFN-gamma(-/-) mice have the ability to both induce and suppress granuloma formation. Induction of granuloma is probably dependent on both T cells and IFN-gamma produced by non-T cells. It is suggested that the role of T cells in granuloma formation is not dependent on their IFN-gamma production.     

IgG isotype distribution of local and systemic immune responses induced by influenza virus infection

The IgG isotype profile of the influenza virus-specific immune response was studied by quantitation of serum antibody (Ab) levels in correlation with the enumeration of antibody-secreting cells (ASC) detected in the lung, spleen, mediastinal lymph nodes (MLN), Peyer's patches and bone marrow (BM). Distincst isotypic patterns for serum Ab and Ab produced by cells present at or close to the site of infection were found after primary or repeated infections. An elevated number of IgM ASC was found after primary challenge in the spleen, lung and MLN. In contrast, the site of IgA and IgG production is restricted to the lung and lymph nodes draining the site of infection. In these organs IgA, IgG2a and IgG1 ASC are found as a result of primary virus infection while viral challenge induces mostly activation of IgA-producing cells and secretion of IgA to the lung lavage. In contrast, the majority (80-90%) of Ab detected in the serum belong to the IgG2a subclass and their serum level is maintained at a high level during the whole period of the response. The relative level of virus-specific serum IgG2a in correlation with the production of IgG2a Ab found predominantly in MLN and lung is highly dependent on the viral dose used for priming or challenge. As IgG2a ASC can be detected at relatively low numbers in the spleen and BM these results suggest that the production of the dominant IgG2a isotype of serum Ab occurs close to the viral challenge site. These data, however, point to distinct isotypic regulation in systemic versus local virus-specific Ab responses.     

Granulocyte‐augmented chemokine production induced by type II collagen containing immune complexes is mediated via TLR4 in rheumatoid arthritis patients

Rheumatoid arthritis (RA) patients with early elevations of antibodies against collagen type II (CII) have a distinct acute onset phenotype, associated with cytokine induction by surface‐bound anti‐CII‐containing immune complexes (ICs) and high C‐reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Polymorphonuclear granulocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) are abundant in the vicinity of CII in RA joints, and both PMN and PBMC reactivity against anti‐CII IC individually relate to early joint destruction and early elevation of CRP and ESR in RA. We searched for CII‐dependent mechanisms that might attract PMNs and PBMCs to RA joints. Human PBMCs and PMNs were stimulated with anti‐CII ICs and control ICs, either individually or in cocultures. Cocultured PMNs and PBMCs stimulated with anti‐CII ICs synergistically augmented production of the chemokines CXCL8, RANTES and MCP‐1, whereas downregulation was seen with control IC. This upregulation was unique to chemokines, as TNF‐α, IL‐1β, and GM‐CSF were downregulated in anti‐CII IC‐stimulated cocultures. The coculture‐associated chemokine upregulation depended on endogenous TLR4 ligand(s) and functionally active PMN enzymes, and was partially mediated by GM‐CSF. As anti‐CII levels peak around the time of RA diagnosis, this mechanism can attract inflammatory cells to joints in early RA and intensify the anti‐CII‐associated acute onset RA phenotype.     

Exacerbation of autoimmune arthritis by copolymer-I through promoting type 1 immune response and autoantibody production

Copolymer-I (COP-I) is an unique immune regulatory polymer that has been shown to suppress experimental autoimmune encephalomyelitis (EAE) and is a treatment option for multiple sclerosis (MS). To investigate whether its immune suppressive effects can be extended to other autoimmune diseases, we treated mice with COP-I during the induction of collagen-induced arthritis (CIA). Our results show that COP-I treatment exacerbated CIA, leading to faster onset, more severe and longer-lasting disease. The mechanisms underlying the exacerbation of CIA by COP-I treatment include enhanced activation and inflammatory cytokine production by autoreactive T cells and elevated production of autoreactive antibodies. In addition, germinal center response was significantly enhanced by COP-I treatment. Thus, great caution should be taken when COP-I is to be used in MS patients with other autoimmune complications or its potential therapeutic effects are to be extended beyond autoimmune demyelinating diseases.     

Exacerbation of autoimmune arthritis by copolymer-I through promoting type 1 immune response and autoantibody production

Copolymer-I (COP-I) is an unique immune regulatory polymer that has been shown to suppress experimental autoimmune encephalomyelitis (EAE) and is a treatment option for multiple sclerosis (MS). To investigate whether its immune suppressive effects can be extended to other autoimmune diseases, we treated mice with COP-I during the induction of collagen-induced arthritis (CIA). Our results show that COP-I treatment exacerbated CIA, leading to faster onset, more severe and longer-lasting disease. The mechanisms underlying the exacerbation of CIA by COP-I treatment include enhanced activation and inflammatory cytokine production by autoreactive T cells and elevated production of autoreactive antibodies. In addition, germinal center response was significantly enhanced by COP-I treatment. Thus, great caution should be taken when COP-I is to be used in MS patients with other autoimmune complications or its potential therapeutic effects are to be extended beyond autoimmune demyelinating diseases.     

In vitro production of interferon-gamma by peripheral blood from patients with Graves'' disease, Hashimoto''s thyroiditis and rheumatoid arthritis.

The production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC), CD4 cells, or CD8 cells in response to interleukin-2 (IL-2) stimulation has been studied; the samples were obtained from 12 healthy control subjects, 19 patients with Graves' disease (10 hyperthyroid and nine euthyroid), 13 patients with Hashimoto's thyroiditis (four hypothyroid and nine euthyroid), and 15 patients with rheumatoid arthritis (11 active and four inactive). A dose of IL-2 (25 U/ml) was utilized to induce IFN-gamma by PBMC from all four groups. The incremental increase in IFN-gamma values (with IL-2 stimulation minus without stimulation) was significantly less in PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis than that in PBMC from control subjects. The values from PBMC in patients with Graves' disease in a euthyroid state were below normal but greater than those from patients with Graves' disease in a hyperthyroid state. The incremental increase in IFN-gamma values from Graves' disease PBMC correlated with the serum TSH values (r = 0.622, P less than 0.01), but not with thyroid autoantibodies (anti-thyroid microsomal antibodies, anti-thyroid microsomal antibodies, nor TSH-binding inhibitory immunoglobulin activities). The incremental increase in IFN-gamma from PBMC from both control subjects and Graves' disease was correlated with that from CD4 cells (r = 0.711, P less than 0.01), but not with that from CD8 cells. The production of IFN-gamma in response to IL-2 from PBMC in Graves' disease correlated inversely with thyroid function, appearing to reflect the very effect of hyperthyroidism in this process. The precise explanation of these phenomena remains unclear. The decreased response of IFN-gamma to IL-2 stimulation by PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis seems to be a non-specific phenomenon occurring in both organ specific autoimmune disease and systemic autoimmune disease. It may be due to a down-regulation in autoimmune disease of CD4 cells in response to IL-2, a decreased level of IL-2 cellular receptors or a decreased receptor affinity, associated increased soluble IL-2 receptors, or a defect of the intra-CD4 cellular IL-2 signal to produce or release IFN-gamma in the conditions studied.     

Immunopathological investigations during the course of arthritis induced in rats by Streptococcus agalactiae

A large single intraperitoneal injection of sonicated cells of Streptococcus agalactiae O90R induced polyarthritis in Wistar rats. The arthritis reaction score was monitored according to redness, edema, severity and deformity of rat ankle and wrist joints. The inflammation of joints was confirmed by radiology and histology. Acute arthritis was initiated within 48 h and the chronic form continued for more than 30 days. Although serum immunoglobulin was elevated within 48 h, anti-streptococcal antibody was detected only at later times (by enzyme-linked immunosorbent assay and Arthus-type hypersensitivity reactions) and neither serum nor splenocytes of arthritic rats were able to transfer disease to susceptible, normal rats. From these observations and the finding of streptococcal antigen in joint macrophages (by immunogold labelling) we conclude that arthritis is related to persistent streptococcal fragments rather than to antibody or immune complexes.     

Murine immune response induced by Leishmania major during the implantation of paraffin tablets

We carried out a model of chronic inflammation using a subcutaneous paraffin tablet in mice experimentally infected with Leishmania major. It was previously reported that the parasite load following paraffin implantation occurred at a peak of 21 days in both BALB/c and C57BL/6 mice. At the present study, we have investigated what cytokines and chemokines are directly related to the parasite load in C57BL/6 mice. All mice were divided in four groups: mice implanted with paraffin tablets; mice experimentally infected with L. major; mice implanted with paraffin tablets and experimentally infected with L. major; and mice submitted only to the surgery were used for the Real-Time Polymerase Chain Reaction (RT-PCR) controls. Fragments of skin tissue and the tissue surrounding the paraffin tablets (inflammatory capsule) were collected for histopathology and RT-PCR studies. By 21 days, a diffuse chronic inflammatory reaction was mainly observed in the deep dermis where macrophages parasitized with Leishmania amastigotes were also found. RT-PCR analysis has shown that BALB/c mice showed strong IL-4 and IL-10 mRNA expression than controls with very little expression of IFN-γ. In contrast, both IFN-γ and IL-10 mRNA was found in higher levels in C57BL/6 animals. Moreover, in C57BL/6 mice the expression of chemokines mRNA of CCL3/MIP-1α was more highly expressed than CCL2/MCP-1. We conclude that the Th1 immune response C57BL/6 did not change to a Th2 response, even though C57BL/6 animals presented higher parasitism than BALB/c mice 21 days after infection and paraffin implantation.     

Lack of activation induced cell death in human T blasts despite CD95L up-regulation: protection from apoptosis by MEK signalling

The generation of effective immunity requires that antigen-specific T cells are activated, clonally expanded and ultimately eliminated by apoptosis. The involvement of CD95-mediated apoptosis in T-cell elimination is well established, but the conditions which regulate the death pathway under normal circumstances are still emerging. Using superantigen-activated human T cells, we found that whilst T-cell receptor (TCR) signalling triggered up-regulation of CD95 ligand (CD95L), the majority of T cells were resistant to apoptosis induction, despite co-expressing high levels of CD95. Resistance was maintained following direct antibody-mediated cross-linking of CD95 and was not confined to early time periods following activation. Our data implicate TCR-derived signals in protection from apoptosis and reveal a role for the mitogen-activated protein (MAP) kinase pathway by use of a MAP kinase kinase (MEK) inhibitor. Collectively these data demonstrate that resistance to activation-induced cell death in human T cells is prolonged rather than transient, is not attributable to a lack of CD95L up-regulation and is due, at least in part, to signalling via the MEK pathway.     

B cell-mediated down-regulation of IFN-gamma and IL-12 production induced during anti-tumor immune responses in the tumor-bearing state

Unfractionated spleen cells taken from tumor-bearing mice contained tumor-primed T cells which produced lymphokines such as IFN-gamma and IL-2 through collaboration with antigen-presenting cells (APC) binding tumor antigens when cultured in vitro. Here, we investigated the regulatory mechanisms underlying IFN-gamma production by T-APC interactions. Elimination of B cells from a splenic population of tumor- bearing mice resulted in enhanced IFN-gamma production. Adding B cells back into cultures down-regulated IFN-gamma production to almost the same levels as those induced by unfractionated spleen cells. IL-2 production was not enhanced by B cell depletion, but rather was significantly suppressed. IFN-gamma-selective up-regulation was due to an enhancement of IL-12 production because IL-12 was detected in B cell- depleted cultures and enhanced IFN-gamma production was prevented by addition of anti-IL-12 mAb or anti-CD40 ligand (CD40L) mAb capable of inhibiting CD40L-induced IL-12 production. These results indicate that B cells interfere with IFN-gamma production induced through interactions between anti-tumor T cells and APC, and this suppressive effect is based on the capacity of CD40+ B cells to down-regulate the CD40L-induced IL-12 production by APC.      

Cell death and cell proliferation during atrophy of the rat parotid gland induced by duct obstruction

Rat parotid gland atrophy after unilateral duct ligation was studied by light and electron microscopy. Death of secretory acinar cells, which took the form of apoptosis, resulted in their complete disappearance within 5 days. The remnants of the dying cells were mostly phagocytosed and degraded by macrophages within the glandular epithelium; a few were taken up by adjoining epithelial cells. The acinar cell deletion was accompanied by increased mitosis of striated and intercalated duct epithelial cells. However, over many weeks, there was enhanced apoptosis of duct cells, which eventually led to marked shortening of intercalated ducts. Apoptosis of capillary endothelial cells was observed and may account for the reduction in the capillary bed known to accompany gland atrophy. The end-stage lesion comprised small numbers of ducts in a condensed stroma. Compensatory hyperplasia, involving proliferation of duct and acinar cells, was demonstrated in the contralateral glands.