Granuloma Formation In Mice Research Articles

Role of CCR4 Ligands, CCL17 and CCL22, During Schistosoma mansoni Egg-Induced Pulmonary Granuloma Formation in Mice

Controversy persists pertaining to the role of CCR4 ligands, namely CCL17 (or thymus and activation regulated chemokine; TARC) and CCL22 (or macrophage-derived chemokine; MDC), in Th2-type cytokine-dominated responses in the lung. Accordingly, the present study addressed the relative role of each of these CC chemokines during an evolving pulmonary granulomatous response elicited by the intrapulmonary embolization of live Schistosoma mansoni eggs into S. mansoni-sensitized mice. CCL22 protein expression peaked at day 4, but CCL17 levels were not increased significantly at any time after egg challenge. CCR4 transcript and protein expression were highest at day 8 after egg embolization and CCR4 protein was prominently expressed in macrophages surrounding S. mansoni eggs. Systemic immunoneutralization of CCL22 from the time of egg injection into S. mansoni-sensitized mice for 8 days significantly decreased CCR4 protein expression, the eosinophil content, the overall size of the egg granuloma, and its hydroxyproline content. Whole lung levels of interferon-gamma were also significantly increased at day 8 in anti-CCL22-treated mice. The systemic immunoneutralization of CCL17 had a lesser effect on all of the granuloma parameters listed above, but this antibody treatment significantly decreased granuloma hydroxyproline content to a greater extent than the anti-CCL22 antibody treatment. In addition, the immunoneutralization of CCL17 significantly increased whole lung levels of interleukin (IL)-4, IL-5, IL-13, transforming growth factor-beta, IL-12, and tumor necrosis factor-alpha at day 8 after egg infusion. Thus, these studies demonstrate a major role for CCL22 and a lesser role for CCL17 during an evolving S. mansoni egg granuloma in the lung.

Effect of Osteopontin Alleles on β-Glucan-Induced Granuloma Formation in the Mouse Liver

The granuloma formation is a host defense response against persistent irritants. Osteopontin is centrally involved in the formation of granulomas. Three osteopontin alleles, designated a, b, and c, have been found in mice. Here we used a murine model of zymosan (beta-glucan)-induced granuloma formation in the liver to determine possible functional differences between the osteopontin alleles in cell-mediated immunity. In contrast to mice with alleles a or c, mice with the allele b was defective in granuloma formation. As detected by mRNA expression, cytokines and chemokines known to be critically involved in granuloma formation were elicited in liver tissue, regardless of the osteopontin allele expressed. Alignment of the deduced amino acid sequences showed that unlike osteopontin c, b differs from a in 11 amino acids. All three osteopontin alleles had normal cell-binding properties. However, only the b allelic form was defective in the induction of cell migration as tested with dendritic cells. In conclusion, generation of a granulomatous response in mice depends critically on the presence of a functional osteopontin allele. Defective granuloma formation in mice with allele b is likely to be because of an impaired chemotactic function of the osteopontin b protein on immunocompetent cells.

Role of AIM in Corynebacterium-induced granuloma formation in mice

Role of AIM in Corynebacterium-induced granuloma formation in mice

Role of AIM in Corynebacterium-induced granuloma formation in mice.

Apoptosis inhibitor expressed by macrophages (AIM) is a murine macrophage-specific protein and belongs to the macrophage scavenger receptor cysteine-rich domain superfamily. AIM has been introduced as the inducer of resistance to thymocyte apoptosis [1]. Because apoptosis of inflammatory cells plays a pivotal role in inflammation [2], we have applied a mouse model to address potential involvement of AIM in the process of granulomatous inflammation in vivo.

AIM Inhibits Apoptosis of T Cells and NKT Cells in Corynebacterium-Induced Granuloma Formation in Mice

Apoptosis inhibitor expressed by macrophages (AIM) inhibits apoptosis of CD4(+)CD8(+) (CD4/CD8) double-positive thymocytes, and supports the viability of these cells on the thymic selection. However, pleiotropic functions of AIM have been suggested. In this study, heat-killed Corynebacterium parvum (C. parvum) was injected into mice carrying the homozygous mutation (AIM(-/-)) and wild-type (AIM(+/+)) mice, to investigate the role of AIM in the formation of hepatic granulomas. In AIM(-/-) mice, the size and the number of hepatic granulomas were larger, and the resorption of granulomas was more delayed than in AIM(+/+) mice. The production of interleukin-12 was more prominent in AIM(-/-) mice than in AIM(+/+) mice. In the liver of AIM(+/+) mice, expression of AIM messenger ribonucleic acid (mRNA) increased after C. parvum injection. In situ hybridization demonstrated that AIM mRNA was expressed in Kupffer cells and exudate macrophages in the liver, especially in granulomas. Larger numbers of T cells and natural killer T (NKT) cells underwent apoptosis in the granulomas of AIM(-/-) mice, suggesting that AIM prevents apoptosis of NKT cells and T cells in C. parvum-induced inflammation. Recombinant AIM (rAIM) protein significantly inhibited apoptosis of NKT cells and T cells obtained from C. parvum-stimulated livers in vitro. These results indicate that AIM functions to induce resistance to apoptosis within NKT cells and T cells, and supports the host defense in granulomatous inflammation.

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.

Chemokine responses in schistosomal antigen-elicited granuloma formation.

Host immune systems have evolved specialized responses to multicellular parasites. This is well represented by the type 2 granulomatous response to Schistosoma mansoni egg antigens, which is an eosinophil-rich inflammatory response mediated by Th2-associated cytokines. Using Ag-bead models of pulmonary granuloma formation in mice, we defined characteristic chemokine (CK) profiles in the granulomatous lungs. Our findings point to a role for C-C chemokine receptor-2 (CCR2) and CCR3 agonists such as monocyte chemotactic proteins (MCPs) 1/CCL2, 3/CCL7 and 5/CCL12 as important participants that are subject to regulation by Th2 cytokines interleukin (IL)-4 and IL-13. CCR4 and CCR8 agonists are also likely contributors. Analysis of CK receptor knockout mice revealed that CCR2 ligands (e.g. MCP-1 and 5) promoted early phase granuloma macrophage accumulation, whereas anti-MCP-3 (CCL7) antibody treatment abrogated eosinophil recruitment. CCR8 knockout mice also demonstrated impaired eosinophil recruitment but this appeared to be related to impaired Th2 cell function. Transcript analysis of CD4+ T cells generated during schistosome granuloma formation failed to show biased CCR8 expression but, having a more limited receptor repertoire, these cells were likely more dependent on CCR8 ligands. Together, these studies indicate an intricate involvement of chemokines in various stages and aspects of schistosomal egg Ag-elicited granuloma formation.

Corynebacterium parvum- andMycobacterium bovisBacillus Calmette and Guerin-Induced Granuloma Formation in Mice Lacking CD4 and CD8

Granuloma formation is a T-cell-dependent inflammatory response that is important in the host defense against intracellular bacteria. The role of CD4 and CD8 molecules in the development ofCorynebacterium parvum- andMycobacterium bovisBacillus Calmette and Guerin (BCG)-induced granulomas was examined in CD4/CD8 knockout (KO) mice. CD4/CD8 KO mice developed a greater granulomatous response to heat-killedC. parvumand heat-killed BCG than did control mice. Thus, granuloma formation is not dependent upon the presence of CD4 and CD8. On the other hand, CD4/CD8 KO mice challenged with live BCG showed initially fewer and smaller granulomas but later more and larger granulomas than control mice. CD4/CD8 KO mice had a greater BCG load than control mice. The absence of CD4 and CD8 therefore impaired the host defense against infection with BCG. αβ T-cells were present in the granulomas of both CD4/CD8 KO and control mice in similar numbers. Also the production of IFN-γ mRNA was similar in the two groups. In conclusion, CD4 and CD8 are not essential to the granulomatous response againstC. parvumand BCG, but contribute to the host defense against live BCG infection.

Role of Macrophage Scavenger Receptors in Hepatic Granuloma Formation in Mice

In mice homozygous for the gene mutation for type I and type II macrophage scavenger receptors (MSR-A), MSR-A-/-, the formation of hepatic granulomas caused by a single intravenous injection of heat-killed Corynebacterium parvum was delayed significantly for 10 days after injection, compared with granuloma formation in wild-type (MSR-A+/+) mice. In the early stage of granuloma formation, numbers of macrophages and their precursor cells were significantly reduced in MSR-A-/- mice compared with MSR-A+/+ mice. In contrast to MSR-A+/+ mice, no expression of monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and interferon-gamma mRNA was observed in MSR-A-/- mice by 3 days after injection. Also in MSR-A-/- mice, uptake of C. parvum by Kupffer cells and monocyte-derived macrophages in the early stage of granuloma formation was lower and elimination of C. parvum from the liver was slower than in MSR-A+/+ mice. In the livers of MSR-A+/+ mice, macrophages and sinusoidal endothelial cells possessed MSR-A, but this was not seen in the livers of MSR-A-/- mice. In both MSR-A-/- and MSR-A+/+ mice, expression of other scavenger receptors was demonstrated. These data suggest that MSR-A deficiency impairs the uptake and elimination of C. parvum by macrophages and delays hepatic granuloma formation, particularly in the early stage.

Granuloma formation activity and mycolic acid composition of mycobacterial cord factor.

We analyzed the mycolic acid composition of trehalose 6,6'-dimycolate (TDM) obtained from Mycobacterium, using thin layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Utilizing TDM, whose structure was confirmed, granuloma formation in mice was investigated. All TDM used exhibited considerable granuloma formation activity in the lung and spleen. In particular, TDM from M. bovis showed the greatest activity and toxicity among mycobacterial TDM. We therefore discussed the relationship between the chemical structure and granuloma-forming activity of TDM, especially in relation to the structure of mycolic acid in TDM.

Kinetic study of eosinophil chemotactic factor production with reference to eosinophilia and granuloma formation in mice infected with Schistosoma japonicum

Kinetic changes of eosinophil chemotactic factor (ECF) production from granulomas, splenic T-cells or mast cells were examined with reference to granuloma formation around newly deposited single eggs in Schistosoma japonicum-infected mice. The peri-ovular granulomas began to appear at around 5 weeks post-infection (p.i.). Their size reached a peak at 6 weeks and then decreased gradually. Up to 8 weeks p.i., eosinophils were the predominant cell type in the granulomas. ECF-release from isolated granulomas paralleled the size of granulomas. Circulating ECF-A, which was assumed to be derived from mast cells, was also detected 6 weeks afterwards in parallel with the level of specific IgE antibody level against egg antigens in the serum. The circulating ECF-A peaked at 8 weeks and decreased after 10 weeks. Spleen cells began to produce ECF specific to bone-marrow eosinophils began at 5 weeks p.i., reached a peak at 6 weeks and then decreased rapidly. On the other hand, the production of ECF specific to eosinophils obtained from the peritoneal cavity began at 6 weeks and decreased rapidly thereafter. These results suggest that various kinds of host-derived ECFs seem to contribute, in one way or an other, to the accumulation of eosinophils in and around granulomatons lesions. The possible role of these ECFs in eosinophil mobilization from the site of production to the inflamed site is discussed.

Endogenous interleukin 12 (IL-12) regulates granuloma formation induced by eggs of Schistosoma mansoni and exogenous IL-12 both inhibits and prophylactically immunizes against egg pathology.

Morbidity in humans infected with Schistosoma mansoni results primarily from the deposition of parasite eggs in portal areas where they induce a granulomatous response. In mice infected with this helminth granuloma formation is a CD4+ T helper (Th) cell-dependent process that is associated with a strong Th2 cytokine response which appears to evolve through a Th0 phase. In this report, we asked whether endogenously synthesized or exogenously induced interferon (IFN)gamma through its suppression of Th2 cell expansion exerts a regulatory role on egg pathology. Depletion of IFN-gamma or natural killer cells resulted in a marked enhancement of granuloma formation around intravenously injected eggs and was associated with increased Th2 and decreased Th1 and interleukin (IL)12 mRNA expression. Similar changes occurred when egg-injected mice were treated with neutralizing monoclonal antibodies specific for IL-12 indicating a role for this cytokine in the regulation of the granulomatous response. In contrast, treatment with exogenous rIL-12 profoundly inhibited primary granuloma formation while increasing IFN-gamma, IL-2, IL-10, and IL-12 pulmonary mRNA levels and suppressing IL-4, IL-5, IL-6, and IL-13 mRNA expression. Cytokine depletion studies indicated that the effects of IL-12 could be attributed primarily to increased IFN-gamma. Importantly, IL-12 also inhibited secondary granuloma formation in mice presensitized with eggs demonstrating a role for the cytokine in reversing established Th2-type responses. Moreover, mice sensitized with eggs in combination with IL-12 to precommit them toward a Th1 response developed only minimal granulomas upon subsequent egg challenge. The latter findings suggest that simultaneous vaccination with antigen plus IL-12 may provide a strategy for the prevention of schistosome egg pathology as well as other diseases stemming from Th2 cytokine production.

Suppression of pulmonary granulomatous inflammation by immunomodulating agents.

We demonstrated previously that macrophages and macrophage-derived cytokines including lymphocyte-activating factors (LAFs) play a critical role in lung granuloma formation in mice and that granulomas sizes correlated with LAF activity in the lesions. In the present study, we examined the effects of D-penicillamine (D-Pc), 2-acetylthiomethyl-3-(4-methyl-benzoyl)propionic acid (KE-298) and dexamethasone (Dex) on dextran bead-induced lung granulomas in mice. KE-298 is a newly synthesized compound containing sulfur (S) similar to D-Pc. Large granulomas developed, which reached peak intensity within 3 days and declined in size thereafter. Aqueous lung extracts of the mice contained high levels of LAF that were correlated with granuloma sizes. The lesions and local LAF activity were inhibited by administration of these agents. The most potent inhibitor was Dex. The suppressive effect of KE-298 was similar to that of D-Pc. These results suggest that suppression of granulomas may be attributed to inhibition of LAF activity/synthesis by these agents.

Genetic control of streptococcus-induced hepatic granulomatous lesions in mice.

Hepatic granulomatous lesions were induced in mice by a single intraperitoneal injection of 3 mg of disrupted Streptococcus pyogenes cell-wall material. Mice carrying the H-2b or H-2k haplotypes were highly susceptible to the induction and three weeks after the injection produced numerous granulomas. In contrast, mice of the H-2d haplotype were resistant and produced only a few hepatic granulomas. Resistance was inherited as a dominant trait and in the backcross generation segregated together with the H-2d phenotype. Testing of the H-2-recombinant mice indicated that the putative gene(s) determining resistance/susceptibility is located to the right of the S and to the left of the D region. This location corresponds to the recently described gene cluster consisting of tumor necrosis factor (TNF) and lymphotoxin genes and several BAT sequences. The known effect of TNF on granuloma formation in mice is consistent with a possible effect of TNF genes, and their variants, on S. pyogenes-inducibility of hepatic granulomas in mice.

Immunogenetic influences on skin granuloma formation in mice

Genetic influence on the development of granulomatous tissue reaction was investigated in C57BL/6 mice. Granulomas developed in the skin of euthymic C57BL/6 mice by transplantation of lyophilized hepatic granulomas were excised and lyophilized. The tissue mass free of parasite egg antigen and living cells was grafted into the skin of euthymic, athymic ( nu nu ), and beige ( bg bg ) C57BL/6 mice. Histological changes at the skin sites were studied weekly by light microscopy, and cells in newly developed granulomas at 6 weeks after grafting were examined by electron microscopy. Granulomatous inflammation occurred in all the variants but morphometric analysis showed that granulomatous inflammation was the most extensive in beige mice and least in athymic mice. The differences in the degree of tissue reaction were also quantified by measuring angiotensin converting enzyme and prolyl endopeptidase. Statistically significant differences among the animals with varying genetic background were confirmed by the marker enzyme activity. The findings confirm that initiation of a granulomatous response does not require T cells but T cell function is important for full expression of the reaction, while NK cell activity seems to suppress granuloma formation.

Mycolic Acid‐Containing Glycolipid as a Possible Virulence Factor of Rhodococcus equi for Mice

By the use of various Rhodococcus equi strains differing in the length of carbon chains of glycolipid, we examined whether the glycolipid, glucose monomycolate, was contributing to the virulence of R. equi for mice. R. equi strains with longer carbon chain mycolic acid showed a higher virulence as determined by lethality and granuloma formation in mice than those with shorter ones. When purified glycolipid was injected into mice, granuloma formation and liver damage were most prominent with the glycolipid having longer carbon chain mycolic acid. Only a representative strain with longer carbon chain mycolic acid persisted in the spleen of mice after intravenous injection, while a strain with shorter carbon chain mycolic acid was readily eliminated. These results suggested that glycolipid was at least one of the virulence factors of R. equi and that the carbon chain length of mycolic acid might be critical in the expression of virulence.

Kupffer Cell Proliferation and Glucan-Induced Granuloma Formation in Mice Depleted of Blood Monocytes by Strontium-89

In mice with prolonged severe monocytopenia induced by selective irradiation of the bone marrow with the bone-seeking isotope 89Sr, the proliferative capacity of Kupffer cells was studied by immunohistochemistry with an anti-mouse macrophage monoclonal antibody, F4/80, ultrastructural peroxidase (PO) cytochemistry, and tritiated thymidine (3HTdR) autoradiography. The number and 3HTdR uptake of Kupffer cells were significantly increased in the splenectomized mice after severe monocytopenia had continued for more than 4 wk, and almost all the Kupffer cells showed a localization pattern of PO activity similar to that of resident macrophages in the liver of normal mice. In the glucan-induced granuloma formation in similar monocytopenic mice, Kupffer cells proliferated, conglomerated, and transformed into epithelioid cells, which fused together to become multinuclear giant cells. These results suggest that Kupffer cells are a self-renewing population by their own cell division and can participate actively in granulomatous inflammations in severely monocytopenic and intact mice.

Parallel antitumor, granuloma-forming and tumor-necrosis-factor-priming activities of mycoloyl glycolipids from Nocardia rubra that differ in carbohydrate moiety: structure-activity relationships.

Multiple intravenous injections (30 micrograms, ten times) in ICR mice of trehalose dimycolate and glucose monomycolate from Nocardia rubra, containing C36-48 mycolic acids, showed a prominent antitumor effect on a subcutaneously implanted sarcoma-180, an allogeneic sarcoma of mice with a significant granuloma formation in lungs, spleen and liver. On the other hand, mycoloyl glycolipids other than glucose monomycolate and trehalose dimycolate, such as mannose or fructose mycolate, showed no significant activity for tumor regression or granuloma formation in mice. Trehalose dimycolate and glucose monomycolate from N. rubra, and glucose monomycolate with C56-60 mycolic acids from Rhodococcus terrae also showed a distinctive priming activity for tumor necrosis factor (TNF), when lipopolysaccharide from Escherichia coli was administered as an eliciting agent. The TNF activity in the sera of mice was abrogated almost completely by anti-(murine TNF alpha) antibody with protein-A-agarose. Again in contrast, mannose and fructose mycolate from N. rubra and glucose monomycolate with C30-34 mycolic acids from Rhodococcus equi did not show such activities in mice. Meth-A, a syngeneic fibrosarcoma of BALB/c mice, was less sensitive to administration of glycolipids than sarcoma-180. These results indicated that the existence of a glucose or trehalose molecule was necessary for the expression of immunomodifying activities among various mycoloyl glycolipids differing in carbohydrate structure. However, since the administration of lipopolysaccharide was essentially required as an eliciting agent for the induction of TNF, while no eliciting agent was required for the antitumor activities, TNF does not seem to contribute directly to the antitumor activities of mycoloyl glycolipids in our systems. There was, however, a parallel structure-activity relationship among granuloma-forming, antitumor and TNF-priming activities, indicating that the structures of both the carbohydrate moiety and the mycoloyl residues influenced an initial step, such as macrophage activation, commonly and profoundly.

Mycolyl glycolipids stimulate macrophages to release a chemotactic factor

We have previously reported that mycolyl glycolipids from Nocardia rubra such as glucose or trehalose mycolates induced granuloma formation in mice [1,2]. The structure of the carbohydrate moiety of the mycolyl glycolipids influenced the granuloma forming activity profoundly. Here, we have examined the macrophage-chemotactic activity in the culture supernatants stimulated with various glycolipids differing in carbohydrate moiety (trehalose 6,6′-dimycolate, or TDM; glucose monomycolate, or GM; mannose monomycolate, or MM; and fructose monomycolate, or FM). A distinctive chemotactic activity was detected with TDM or GM, but little or none with MM or FM.

The role monokines in granuloma formation in mice: The ability of interleukin 1 and tumor necrosis factor-α to induce lung granulomas

Granulomatous inflammation is associated with many significant human diseases, including tuberculosis, leprosy, sarcoidosis, parasite infection, and berylliosis. Very little is known about the basic mechanism of this type of inflammation. In the present study, we showed that pulmonary granulomas were induced in mice by the intractracheal injection of agarose beads coupled to recombinant interleukin 1 (IL-1) or tumor necrosis factor-α (TNF-α). Histologically, the bulk of granulomas was composed of macrophages and their derivatives. In contrast, the inflammatory reactions induced by beads coupled to either recombinant IL-2 or murine interferon-γ were considerably smaller than those induced by beads coupled to monokines. These results suggest that macrophages and monokines such as IL-1 and TNF-α, but not T cell-derived lymphokines, play an essential role in granuloma formation.