Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.

Synchronized pulmonary granulomas (GRs) were induced in presensitized mice by intravenous embolization of polymer beads bound with purified protein derivative (PPD) of Mycobacteria tuberculosis or soluble antigens derived from Schistosoma mansoni eggs (SEA). Uncoated beads served as a foreign body control (CON). Antigen-coated beads elicited GRs with characteristic epithelioid macrophages and multinucleate giant cells by 4 days after embolization. Unlike PPD GR, SEA bead lesions contained eosinophils, whereas CON beads elicited only a limited mononuclear infiltrate. GRs and draining lymph nodes (LN) were assessed on days 2, 4, and 8 for Th1-(interleukin-2 [IL-2], interferon-gamma[IFN] and Th2-type (IL-4, IL-5, and IL-10) cytokines. CON GR produced only a small amount of IFN-gamma on day 2 and failed to induce a significant response in draining LN. In contrast, both PPD and SEA antigen-coated beads induced reactive lymphoid hyperplasia but differed greatly in local and regional cytokine profiles. PPD GR produced IFN-gamma on day 2 and the draining LN produced predominantly Th1 cytokines on days 2 and 4. In contrast, SEA beads GRs were dominated by Th2 cytokines. The corresponding LN produced IL-2 and IL-4 on day 2; IL-2, IL-4, IFN-gamma, and IL-10 on day 4; then IL-2, IFN-gamma, and IL-4 on day 8, probably reflecting maturational changes of T cells. Macrophages (MP) from bead GR also showed different patterns of IL-6 and tumor necrosis factor (TNF) production. Compared with CON GR, MPs from PPD GR were weak sources of IL-6, whereas those of SEA GR showed enhanced and accelerated production. In contrast, MP of PPD GR had augmented TNF-producing capacity, whereas those of SEA GR showed delayed TNF production. In vivo depletion of TNF, respectively, caused 40 and 10% decreases in PPD GR and SEA GR but had no effect on CON GR area, indicating that TNF contributed to a greater degree to the PPD response. These data show that depending on the inciting agent, GR can be mediated by different cytokines. Characterization of inflammatory lesions by cytokine profiles should allow design of more rational therapeutic interventions.     

AMD3465, a novel CXCR4 receptor antagonist, abrogates schistosomal antigen-elicited (type-2) pulmonary granuloma formation

CXCR4 is a major receptor for CXCL12 and is known to participate in multiple physiological systems. The present study tested a second generation CXCR4 antagonist, AMD3465, for effects on highly defined models of Th1- and Th2-cell-mediated hypersensitivity-type pulmonary granuloma formation. Type-1 and type-2 granulomas were induced, respectively, by intravenous challenge of sensitized CBA/J mice with Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg antigen-coated beads. Before challenge, mice were implanted with osmotic pumps releasing AMD3465 at 5 microg/hour (6 mg/kg/day). Compared to vehicle, AMD3465 had minimal effect on type-1 inflammation or cytokine responses in draining lymph nodes, but the type-2 inflammation was significantly abrogated with reductions in lesion size and eosinophil content as well as abrogated interleukin (IL)-5, IL-10, and IL-13 cytokine production in draining lymph nodes. The biased effect of AMD3465 correlated with greater CXCR4 ligand expression in the type-2 model. Treatment during a primary response impaired lymph node IL-2 production after both Mycobacteria bovis purified protein derivative and Schistosoma mansoni egg antigen challenge indicating an unbiased effect during immune induction. In summary, CXCR4 blockade inhibited eosinophil recruitment during type-2 granuloma formation and interfered with primary and secondary T-cell activation events in lymphoid tissue, suggesting potential therapeutic application for chronic hypersensitivity diseases.     

LIM‐only protein FHL2 regulates experimental pulmonary Schistosoma mansoni egg granuloma formation

LIM‐only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL‐4 and IL‐10. FHL2‐knockout (FHL2‐KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti‐inflammatory M2 phenotype following IL‐4 treatment. Furthermore, thioglycollate‐induced migration of macrophages and B cells is enhanced in FHL2‐KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2‐KO mice were challenged with Schistosoma mansoni eggs. FHL2‐KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2‐KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung.     

Cyclophosphamide affects the dynamics of granuloma formation in experimental visceral leishmaniasis

We observed histopathological and ultrastructural hepatic changes following the intracardiac inoculation ofLeishmania donovani amastigotes into inbred LHC hamsters (group I). Since granuloma formation is known to be T-cell-dependent, we also examined infected hamsters under cyclophosphamide immunosuppressive treatment (group ICy) and evaluated the production of interleukin-2 (IL-2) by their cells. Group I showed more intense hepatocyte and endothelial cell clasmatosis as well as hepatocyte degeneration and necrosis, deposits of connective tissue fibers, granulomas with multinucleated giant cells (MGCs) of foreign-body and Langhans' types and reduced production of IL-2 by spleen cells. In contrast, group ICy hamsters exhibited larger eosinophil and lymphocyte populations within sinusoids and peri-sinusoidal areas but showed no MGCs in granulomas. A striking decline in IL-2 production was noted. These results suggest that cyclophosphamide induces a delay in the natural evolution ofL. donovani-induced granulomatous hepatic inflammation.     

Role for matrix metalloproteinase 9 in granuloma formation during pulmonary Mycobacterium tuberculosis infection

Recent studies have shown that matrix metalloproteinases (MMPs) are induced by Mycobacterium tuberculosis during pulmonary infection. Here, expression of MMP-9 during pulmonary M. tuberculosis infection was characterized to determine whether its production correlated with disease resistance in vivo and to determine what role, if any, MMP-9 might have in granuloma formation. Following aerosol infection with M. tuberculosis, dissemination of bacilli occurred earlier in the C57BL/6 resistant mouse strain than in the susceptible CBA/J strain, as was evident from an increased number of bacteria in the blood, spleen, and liver at day 14 after infection. In addition, early dissemination of the bacilli was associated with early induction of protective immunity as assessed from gamma interferon levels. Nonspecific blocking of MMPs in C57BL/6 mice early during infection reduced hematogenous spread of the bacilli, suggesting that MMPs indeed play a role in facilitating dissemination, likely via extracellular matrix degradation. The concentration of active MMP-9, specifically, was greater in the lungs of C57BL/6 mice than in those of the CBA/J mice at day 28, thereby suggesting that MMP-9 is not one of the MMPs directly involved in promoting early dissemination of M. tuberculosis. Instead, however, histological lung sections and flow cytometric analysis of lung cells from MMP-9-knockout mice showed that MMP-9 is involved in macrophage recruitment and granuloma development. These combined data support the idea that early MMP activity is an essential component of resistance to pulmonary mycobacterial infection and that MMP-9, specifically, is required for recruitment of macrophages and tissue remodeling to allow for the formation of tight, well-organized granulomas.     

Interleukin-13 fusion cytotoxin arrests Schistosoma mansoni egg-induced pulmonary granuloma formation in mice

Schistosoma mansoni egg-induced lung pathology requires the actions of interleukin (IL)-4 and IL-13. Because receptors for IL-4 and IL-13 share chains, we examined the effect of a fusion protein comprised of IL-13 and Pseudomonas exotoxin (IL13-PE) on the development of pulmonary granulomas in mice. At day 8 after an intravenous injection of live S. mansoni eggs, whole lung samples from IL13-PE-treated mice exhibited significantly lower IL-4 and IL-13 gene expression, smaller granulomas, decreased collagen levels, and increased IL-13 receptor alpha2 gene expression compared to controls. The therapeutic effects of IL13-PE were also observed at day 16 despite the termination of IL13-PE treatment at day 8. These studies demonstrate that targeting IL-4- and IL-13- responsive cells with IL13-PE effectively arrests S. mansoni egg granuloma formation.     

Local macrophage proliferation in multinucleated giant cell and granuloma formation in experimental Goodpasture's syndrome.

Granuloma is a specialized form of inflammatory reaction featuring focal macrophage and T-cell accumulation and multinucleated giant cell formation. It is widely held that macrophage accumulation within granulomatous lesions results from recruitment of blood monocytes, whereas proliferation of monocyte/macrophages makes little contribution to this process. The present study of macrophage proliferation within immunologically induced granulomas in rat experimental Goodpasture's syndrome challenges the conventional view. In this disease, granulomatous lesions in the kidney and lung contained 60 to 70% macrophages of an ED1+ED2-ED3-blood monocyte phenotype. However, double immunohistochemistry showed that up to 75% of ED1+ macrophages within granulomatous lesions were proliferating on the basis of proliferating cell nuclear antigen expression and bromodeoxyuridine incorporation. In contrast, no proliferating cell nuclear antigen expression or bromodeoxyuridine incorporations was detected in blood monocytes, indicating that proliferation of ED1+ED2-ED3- cells was a localized event within granulomatous lesions. A second finding of note was that almost all ( > 95%) nuclei within multinucleated giant cells were positive for proliferating cell nuclear antigen, but these nuclei lacked bromodeoxyuridine incorporation. This suggests a novel mechanism of multinucleated giant cell formation involving fusion of macrophages in G1 phase, which then halts progression into S phase of the cell cycle. In conclusion, this study has found that local macrophage proliferation plays an important role in the pathogenesis of granuloma formation.     

Effect of antimacrophage serum on dermal tuberculin sensitivity and allergic pulmonary granuloma formation in rabbits

Anti-rabbit alveolar macrophage goat serum (AMS), adsorbed of hemolysins and hemagglutinins, exhibited complement-dependent cytotoxicity for the following rabbit target cells: alveolar macrophages, thymocytes, and kidney (RK-13) cells. The intravenous injection of AMS, sufficient to reduce markedly the dermal tuberculin reaction in BCG-sensitized rabbits, did not suppress the development of BCG-induced chronic and accelerated pulmonary granulomas. These observations are discussed in relation to delayed sensitivity, allergic granuloma formation, and cell-associated immunity.     

Macrophage and T lymphocyte apoptosis during experimental pulmonary tuberculosis: their relationship to mycobacterial virulence

The kinetics of macrophage and T lymphocyte apoptosis were determined in a well-characterized mouse model of pulmonary tuberculosis, comparing strains of intermediate (H37Rv) and high virulence (Beijing strain, code 9501000). Both strains induced a high percentage of apoptotic activated macrophages at days 1 and 3 post infection, although this was twofold lower in Beijing-infected mice. Progressive pneumonia started at day 14 (Beijing) or 21 (H37Rv) post infection. Pneumonic areas contained numerous macrophages with vacuolated cytoplasm (VM). In H37Rv infection few VM were apoptotic (8.7% at day 60), and the percentage was even lower in Beijing infection (1.4% at day 28). A high percentage of VM expressed the anti-apoptotic molecule Bcl-2 (H37Rv, 83%; Beijing, 95%). Both strains induced a progressive increase of apoptotic Th1 lymphocytes, peaking at day 60 in H37Rv infection, or 28 in Beijing infection. The peak was twofold higher in the latter. VM had strong FasL immunostaining, and confocal microscopy showed numerous apoptotic Th1 cells closely associated with them, suggesting that VM might induce apoptosis of Th1 cells. These results support the hypothesis that apoptosis of macrophages is associated with protection, while apoptosis of Th1 cells favors disease progression, and is related to the virulence of the mycobacterial strain.     

Fibronectin and its role in granuloma formation

Fibronectin (F) is a high-molecular-weight glycoprotein widely presented on the cell surface of fibroblasts, monocytes, endothelial cells and several types of other cells. It is also available in the extracellular matrix of connective tissue and in plasma. Active biologically, F is involved in cell-to-cell and cell-to-substrate adhesion, migration and differentiation of cells, maintenance of cellular structure, wound healing, blood coagulation and opsonic function. In addition, F has a special affinity to collagen, heparin, fibrin and fibrinogen. The above properties suggest F relevance to evolution of productive inflammation and granuloma development. The studies of F presentation, distribution, localization, concentration, function and influence on cell transformation throughout different stages of granuloma evolution can be helpful in elucidation of so far obscure traits of granuloma histogenesis.     

Direct evidence for granuloma-inducing activity of interleukin-1. Induction of experimental pulmonary granuloma formation in mice by interleukin-1-coupled beads.

Pulmonary granulomas were induced in BALB/c mice by the intratracheal injection of Sephadex G-50 and latex beads. Very large granulomas developed around Sephadex G-50 beads. Minimal inflammation was produced in mice given latex beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in mice by Sephadex G-50 beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) activity. IL-1 activity in the extracts correlated with granuloma size. In a subsequent step, large granulomas were induced by the intratracheal injection of Sepharose 4B beads coupled to fractions of the extracts containing IL-1 activity (ie, granuloma-derived IL-1) prepared from Sephadex G-50-induced granulomatous lungs. In addition, large granulomas were induced by the intratracheal injection of recombinant IL-1-coated Sepharose 4B beads. In contrast, very small granulomas were seen when IL-2-coated or plain Sepharose 4B beads were injected into mice. These results indicate that IL-1 participates in the induction and/or expression of granulomas.     

Cryptococcus neoformans pulmonary granuloma formation is associated with matrix metalloproteinase-2 expression.

The aim of this study was to investigate matrix metalloproteinase (MMP) expression during the immune response to pulmonary Cryptococcus neoformans (Cne) infection. The immune response generated in C.B-17 and C57BL/6 mice to pulmonary Cne infection has previously been characterized as type 1 and type 2, respectively, differing in the cytokines produced and leukocytes recruited during infection, influencing the extent of Cne clearance from the lung. The focus of this study was to examine changes in expression of MMP-2 and tissue inhibitor of metalloproteinase (TIMP)-2 in the lungs of Cne-infected mice during the two types (type 1 vs. type 2) of responses. C.B-17 mice that formed well-defined granulomas had elevated levels of pulmonary MMP-2 mRNA early during infection. C57BL/6 mice that had poorly defined cellular aggregates did not express detectable levels of pulmonary MMP-2 mRNA until later in the infection. Specific expression of MMP/TIMP was correlated with the type of immune response present, resolution of Cne infection and the resulting lung pathology.     

Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.