Immune inflammation in newly developing facsimile synovia

The injection of air into the subcutaneous tissue of the back in rodents has previously been shown to induce a lining which closely resembles synovial lining tissue. Air pouches of different ages represent the developmental stages of such tissue. We have found that pouches of 1 day in age induced in animals previously sensitised with bovine serum albumin (BSA) respond with low numbers of infiltrating leucocytes and a small fluid exudate volume 4 h after challenge into the pouch with BSA. In contrast, 3- and 6-day-old pouch tissue when challenged with antigen responds with a greater influx of leucocytes and a larger fluid exudate. These findings suggest that the constituent make-up of the air pouch-lining tissue is important for the full expression of the inflammatory response.     

Changes in histamine and prostaglandins in acute inflammatory air pouch in mice

The changes in histamine and prostaglandins (PGE₂ and PGF) in carrageenan-induced acute inflammation were studied in 6 day old air pouches in mice. A significant elevation of exudate histamine was found 1 hour after the carrageenan injection. Highest vascular permeability at the site of inflammation was also found at 1 hour. Both PGE₂ and PGF showed steady increases in the pouch exudate and reached significantly higher levels at 24 hours. The present findings thus suggest that histamine is involved in the early phase of carrageenan-induced inflammation by enhancing vascular permeability. The increases in PGE₂ and PGF appear to be closely correlated with the increased exudate cell accumulation. This leads us to suggest that they are likely to be involved in the exudate cell activity rather than in enhancing the vascular permeability which was found to decrease at 4 hours after the carrageenan injection.     

The immune response to pertussis in the 6-day air pouch: a model of chronic synovitis

We have developed a model of prolonged immunological inflammation in the rat which has a structural resemblance to the synovial changes in rheumatoid arthritis. Pertussis vaccine was injected into 6-day-old subcutaneous air pouches in animals previously sensitized with pertussis vaccine. The resulting inflammatory response persisted up to 30 days. Examination of exudates showed a wave of polymorphonuclear leucocytes over a 13-day period followed by a mononuclear cell predominance up to 30 days. Histologically, an early polymorphonuclear cell infiltration was followed by the formation of a lining layer of large eosinophilic mononuclear cells, together with deep collections of lymphocytes and plasma cells. Concentrations of the acute-phase reactant alpha 1-glycoprotein, in both serum and exudate, peaked at 3 days. This suggests that the local production of interleukin I in this type of tissue reaction is more closely related to the acute inflammatory phase than to more chronic interactions between monocyte derived cells and lymphocytes.     

Changes in corticosterone levels under different degrees of acute inflammation in mice

Carrageenan of different concentrations was injected into the 6-day-old air pouch in mice. It was found that 12 mg carrageenan caused a significant increase of plasma and exudate corticosterone levels at 24 h, while 1 and 3 mg carrageenan could only induce a significant increase of exudate corticosterone at 4 h. Elevation of corticosterone in both plasma and inflammatory exudate appeared to be correlated, suggesting that the exudate corticosterone was derived from the blood circulation. Injection of exogenous histamine and PGE₂ into the air pouch induced a significant increase in exudate levels of corticosterone. However, plasma corticosterone increased significantly only after histamine administration, although a slight increase was observed in those injected with PGE₂. These findings thus suggest that endogenous histamine and PGE₂ which are released during carrageenan-induced acute inflammation, as shown in our previous work, might be responsible for the increase of corticosterone in both plasma and inflammatory exudate.     

The immune response to pertussis in the 6-day air pouch: a model of chronic synovitis.

We have developed a model of prolonged immunological inflammation in the rat which has a structural resemblance to the synovial changes in rheumatoid arthritis. Pertussis vaccine was injected into 6-day-old subcutaneous air pouches in animals previously sensitized with pertussis vaccine. The resulting inflammatory response persisted up to 30 days. Examination of exudates showed a wave of polymorphonuclear leucocytes over a 13-day period followed by a mononuclear cell predominance up to 30 days. Histologically, an early polymorphonuclear cell infiltration was followed by the formation of a lining layer of large eosinophilic mononuclear cells, together with deep collections of lymphocytes and plasma cells. Concentrations of the acute-phase reactant alpha 1-glycoprotein, in both serum and exudate, peaked at 3 days. This suggests that the local production of interleukin I in this type of tissue reaction is more closely related to the acute inflammatory phase than to more chronic interactions between monocyte derived cells and lymphocytes.     

Analysis of histamine-producing cells at the late phase of allergic inflammation in rats

To identify histamine-producing cells at the late phase of allergic inflammation, the expression of L-histidine decarboxylase (HDC) was examined in the infiltrating leucocytes in the inflammatory locus. HDC activity and HDC mRNA levels in the infiltrating leucocytes in the pouch fluid of the immunized rats (that were injected with the antigen solution into the air pouch) were increased compared with those in the infiltrating leucocytes of the non-immunized rats. When infiltrating leucocytes collected 8 hr after antigen injection were cultured, histamine production by the cells from the immunized rats was higher than that from the non-immunized rats. In situ hybridization of HDC mRNA revealed that almost all the infiltrating leucocytes of the immunized rats, 4 hr after injection of the antigen, expressed HDC mRNA with high intensity, while those of the non-immunized rats showed only a weak intensity of HDC mRNA. In the immunized rats, approximately 90% of leucocytes infiltrating in the pouch fluid at 4 hr were neutrophils and 8% were monocytes/macrophages. Neither mast cells nor basophils were detected in the infiltrating leucocytes. When rat peritoneal neutrophils were incubated in the presence of 12-O-tetradecanoylphorbol 13-acetate, histamine production was significantly increased. These findings suggest that the leucocytes, mainly neutrophils, infiltrating at the inflammatory locus are responsible for histamine production at the late phase of allergic inflammation.     

Enhancement of neutrophil infiltration in histidine decarboxylase-deficient mice

The roles of histamine in the anaphylactic increase in vascular permeability and leucocyte infiltration were analysed in an air pouch-type allergic inflammation model in histidine decarboxylase-deficient (HDC^−/−) mice and wild-type mice. In the immunized wild-type mice, histamine content in the pouch fluid and vascular permeability in the anaphylaxis phase were increased by injection of the antigen solution into the air pouch. However, in the immunized HDC^−/− mice, the antigen challenge did not increase histamine content in the pouch fluid and vascular permeability in the anaphylaxis phase. Number of leucocytes (more than 83% are neutrophils) in the pouch fluid 4–24 hr after the antigen challenge in the HDC^−/− mice was significantly higher than that in the wild-type mice. Simultaneous injection of histamine with the antigen solution into the air pouch of the immunized HDC^−/− mice reduced the antigen-induced leucocyte infiltration at 4 hr. Simultaneous injection of the H2 antagonist cimetidine but not the H1 antagonist pyrilamine with the antigen solution into the air pouch of the immunized wild-type mice further increased leucocyte infiltration at 4 hr. The levels of macrophage inflammatory protein-2 at 2 hr and of tumour necrosis factor-α at 4 hr in the pouch fluid of the HDC^−/− mice were significantly higher than those of the wild-type mice. These findings indicate that histamine plays significant roles not only in the anaphylactic increase in vascular permeability via H1 receptors but also in the negative regulation of neutrophil infiltration via H2 receptors in allergic inflammation.     

An intravital microscopic model for mast cell-dependent inflammation in the hamster cheek pouch

Topical antigen challenge in cheek pouches of immunized hamsters led to an acute inflammatory reaction which was characterized by intravital microscopy. The response consisted of short-lasting arteriolar spasm, followed by leakage of plasma, vasodilation, and accumulation of leucocytes. Several observations indicated that the reaction was due to mast cell activation. Thus, a very similar inflammatory response was seen after challenge with compound 48/80, and both antigen and compound 48/80 degranulated the numerous mast cells present in the cheek pouch. In addition, fluorescein-labelled antigen bound specifically to mast cells in cheek pouches of immunized animals, also suggesting the presence of mast cell-fixed antigen-specific antibodies, possibly immunoglobulin E. However, although antigen and compound 48/80 caused similar microvascular responses, cross-desensitization experiments indicated that the two stimuli activated mast cells via different mechanisms. The histamine antagonist mepyramine, which abolished plasma leakage induced by exogenous histamine, substantially inhibited the increase of microvascular permeability evoked by antigen or compound 48/80, but did not appear to affect the vasospasm and leucocyte accumulation. It is concluded that the hamster cheek pouch may be a most useful tool for investigation of dynamic microvascular events during allergic mast cell-dependent inflammation.     

Antigen handling in antigen-induced arthritis in mice: an autoradiographic and immunofluorescence study using whole joint sections.

Antigen localization after intraarticular antigen injection was studied in immune and nonimmune mice using autoradiographic and immunofluorescence techniques on whole joint sections. After intraarticular injection of radiolabeled methylated bovine serum albumin (125I-mBSA) in immune mice, labeling in the synovium and synovial exudate diminished rapidly, apart from some deposits in fibrinlike material present in the joint cavity. Long-term antigen retention was found in avascular and hypovascular structures lining the joint cavity, albeit not along the whole surface; eg, labeling remained present at the edges of the femoral condyle hyaline cartilage but not at the central weight-bearing region; long-term retention at ligaments was only found at the insertion sites. Immunofluorescence data in immune animals showed antigen retention together with the presence of immunoglobulins and complement, indicating that antigen is retained at least in part in the form of immune complexes. Nonimmune mice showed even higher long-term antigen retention than immune animals, probably related to physico-chemical properties of the antigen enabling nonimmune binding to articular structures, but also indicating that the presence of joint inflammation in the immune animals enhances antigen clearance. Histologic examination of the ligaments and patellar cartilage of immune mice did reveal that long-term antigen retention was not anatomically related to nearby inflammation or to local tissue damage. The importance of long-term antigen retention for the chronicity of arthritis may lie in the leakage of small amounts of this antigen to joint compartments where it does behave as an inflammatory stimulus; it may further be that it renders the joint a specifically hypersensitive area.     

Effect of carrageenan-induced acute inflammation on corticosterone levels in mice

Changes in corticosterone levels in plasma and inflammatory exudase were studied in the 6-day-old air pouch of mice. The pouch inflammation in the test group was induced by the injection of carrageenan prepared in physiological saline while the control received only the physiological saline. The results show that exudate and plasma of both groups showed a rapid rise in corticosterone as measured after 30 min and this early rise was probably due to the resulting effect of the ether used during the injection of irritant or vehicle. In contrast, corticosterone levels in the inflammatory exudate of the test group increased with time, reaching a peak at 24 hours after the carrageenan injection. The increased corticosterone levels in the inflammatory exudate appeared to be closely correlated with the increased exudate cell accumulation. This suggests that the increased accumulation of exudate corticosterone in the pouch might play an important role at the inflammatory site by modulating intensity of the inflammatory reactivity caused by the irritant.     

Increased inflammatory reactivity in newly formed lining tissue

The air pouch has been shown to provide a convenient model for studying the behaviour of synovial lining tissue. Air pouches of different ages were used to study the reactivity of newly developing lining tissue towards irritants known to cause inflammation. Pouches of 1 day in age were relatively inert in their reactivity as judged by the number of cells and volume of the exudate accumulating in the pouch. In contrast, 3-day-old pouches responded to a much greater extent, and 6-day-old pouches were highly responsive with a further increase in cell numbers and fluid volume. The different responses of 1-, 3- and 6-day-old pouches could be explained by (a) developing vascularity of the pouch, (b) formation of an organised lining of phagocytic cells, or (c) an increasingly organised mechanical barrier that retains the irritant and products of the inflammatory response. These studies of air pouch lining development permit a dissection of those components necessary for inflammatory reactivity of a lining tissue and may help explain the sensitivity of synovium to chronic inflammation.     

Thermal injury induces impaired function in polymorphonuclear neutrophil granulocytes and reduced control of burn wound infection

Severe thermal injury induces immunosuppression, involving all parts of the immune system, especially when large fractions of the total body surface area are affected. An animal model was established to characterize the burn‐induced immunosuppression. In our novel mouse model a 6% third‐degree burn injury was induced in mice with a hot‐air blower. The third‐degree burn was confirmed histologically. The mice were allocated into five groups: control, shave, burn, infection and burn infection group. At 48 h, a decline in the concentration of peripheral blood leucocytes was observed in the group of mice with burn wound. The reduction was ascribed to the decline in concentration of polymorphonuclear neutrophil leucocytes and monocytes. When infecting the skin with Pseudomonas aeruginosa, a dissemination of bacteria was observed only in the burn wound group. Histological characterization of the skin showed a more polymorphonuclear neutrophil granulocytes (PMNs)‐dominated inflammation in the group of mice with infected burn wound compared with the with burn wound group. In contrast, a higher degree of inflammation was observed in the burn wound group compared with the group of mice with infected burn wound. Furthermore, the oxidative burst and the phagocytic capacity of the PMNs were reduced in the group of mice with burn wound. Using this novel mouse model of thermal injury a decline of peripheral leucocytes was observed, whereas the increased local inflammatory response at the site of infection showed reduced capacity to contain and eliminate the infection.     

Therapeutic Potential of the Proteasome Inhibitor Bortezomib on Titanium Particle-Induced Inflammation in a Murine Model

Wear particle-induced aseptic loosening has been recognized as a harmful inflammatory process that jeopardizes the longevity of total joint replacement. The proteasome controls the activation of NF-κB and subsequent inflammatory mediators, such as TNF-α and IL-1β; thus, we investigated whether proteasome inhibition can ameliorate wear particle-induced inflammation in a murine model. A total of 48 BALB/C mice were divided into four groups. Titanium (Ti) particles were injected into the established air pouches of all mice (except negative controls) to provoke inflammation, and then 0.1 or 0.5 mg/kg of Bortezomib (Bzb, a proteasome inhibitor) was administered to ameliorate the inflammation response, while air pouches without Bzb administration were used as loading controls. The air pouches were harvested 2 or 7 days after Bzb injection for molecular and histological analyses. Inflammation responses in the air pouch tissues of Bzb treatment groups are lower than those in the Ti-stimulated group, and this occurs in a dose-dependent manner. Bzb can significantly attenuate the severity of Ti-induced inflammation in air pouches.     

Infection of artificial air pouches in the connective tissue of mice with Neisseria gonorrhoeae.

Artificial air pouches in the connective tissue of mice were evaluated as a means of studying Neisseria gonorrhoeae infections. Animals inoculated with type-1 N. gonorrhoeae cells developed an infection characterised by infiltration of polymorphonuclear leucocytes. Viable cocci could be recovered from the air pouches for up to 10 days after infection and intracellular cocci were evident in electronmicrographs within connective-tissue fibroblasts for at least 35 days, indicating that a persistent infection had been established. The mouse air pouch should be of value in the study of gonococcal and other infections.     

Passive Transfer of Delayed Hypersensitivity to Blastomyces dermatitidis Between Mice

This study further characterized the delayed hypersensitivity state induced in animals by Blastomyces dermatitidis exposure. Passive transfer of delayed hypersensitivity by transfer of cells and inhibition of migration of peritoneal exudate cells were studied, using sensitized mice of two inbred strains. Donor mice were subcutaneously inoculated with viable B. dermatitidis yeast cells. After 15 days, spleen cells or serum from these animals were injected intravenously into normal recipients of the same strain. After 24 h these mice were footpad tested with killed B. dermatitidis yeast cell antigen. Mice receiving spleen cells from sensitized animals had a significant increase in footpad thickness 24 to 48 h after testing. Those receiving only serum remained negative. Migration of peritoneal exudate cells from blastomyces-sensitive donor mice was inhibited by presence of blastomycin but not by mycobacterial antigen. Neither blastomyces-sensitive nor control animals reacted to footpad or migration inhibition testing with mycobacterial antigen.     

Increase in Histamine Production by Inflammatory Exudate in the Chronic Phase of Allergic Inflammation in Rats

In the air pouch-type allergic inflammation in rats, we reported that a sustained histamine production in the late phase is induced by a cytokine-like factor, named histamine-production-increasing factor (HPIF) (1). Recently, we found another type of histamine-production-increasing factor in the pouch fluid at the chronic phase of air pouch-type allergic inflammation. Although it did not increase histamine production by itself, it enhanced the HPIF-induced histamine production by rat bone marrow cells. It also increased GM-CSF-induced histamine production. The activity of this factor increased time-dependently from 3 to 7 days after the antigen challenge. Injection of the 5 day pouch fluid sample containing this factor into the pouch 4 h after the antigen challenge increased histamine contents in the pouch fluid at 24 h, indicating that this factor enhances HPIF-induced histamine production in vivo. Biochemical analysis of the 5 day pouch fluid sample indicated that this factor is a heat-labile and trypsin-sensitive protein of which pI value and molecular weight are 7–8 and about 100 kDa, respectively.     

Electrical charge of the antigen determines its localization in the mouse knee joint. Deep penetration of cationic BSA in hyaline articular cartilage.

Intraarticular injection of cationic bovine serum albumin (BSA) induces a chronic arthritis in immunized mice, whereas the negatively charged native BSA fails to cause a protracted joint inflammation. In this study the authors examined the role of antigenic charge as a determinant of antigen retention and exact localization within the knee joint. Immune and nonimmune mice received an intraarticular injection of either radiolabeled native BSA (125I-BSA) or charge-modified BSA rendered cationic by amidation (aBSA), and autoradiographs were prepared of whole joint sections at various days after injection. As has been shown in the rabbit, the retention of the negatively charged native BSA is largely dependent upon the presence of antibodies. In nonimmune mice the radiolabeled antigen was hardly detectable after Day 1. In immune mice antibody-mediated retention of BSA was found in the ligaments and fibrous cartilage structures of the joint but appeared to be absent at the hyaline cartilage. In contrast, large amounts of the cationic aBSA were retained at all collagenous structures of the joint, the most striking observation being the deep penetration in the dense hyaline cartilage. This was found both in immune and nonimmune mice, which indicates that the deep penetration was not due to cartilage damage occurring under inflammatory conditions. With different dosages of aBSA it was found that the presence of antibodies may modulate the retention pattern in immune mice. Deep diffuse penetration into the dense hyaline cartilage, together with some surface labeling, was observed after injection of a high dose (60 micrograms), whereas mere surface labeling was found with the low dose (6 micrograms). Distinct superficial labeling was not seen in nonimmune mice, which suggests that this pattern represents immune complex formation at the cartilage surface. Immunofluorescence studies on undecalcified whole joint sections confirmed the deep penetration of the cationic antigen and supported the presence of immune complexes at the cartilage surface, because intense complement and Ig staining was detectable at this site. Our data indicate that antigenic charge determines the antigen retention in the joint both quantitatively and qualitatively. Negatively charged native BSA has no affinity for cartilage, high amounts of antibodies are needed for its retention in the joint, retention by this immune complex formation is largely restricted to the loose collagenous tissues, and the capacity to retain anionic antigen in the joint is therefore low.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potentiation of IgE-mediated cutaneous reactivity and blood leucocyte histamine release by deuterium oxide.

A previous study (Gillespie & Lichtenstein, 1972) demonstrated that there was potentiation of histamine release from human peripheral blood leucocytes following exposure to antigin or anti-IgE in deuterium oxide (D2O). The current study confirms the results with human leucocytes and indicates that the degree of histamine release due to anti-IgE or its potentiation by D2O appeared independent of the serum IgE concentration of the cell donor. Further studies demonstrated that the peripheral blood leucocytes from monkeys with a sufficient degree of IgE-mediated reactivity to Ascaris antigen-released histamine following exposure to that antigen. This leucocyte histamine release occurs in animals with immediate-type cutaneous and respiratory reactivity following challenge with this antigen. Peripheral blood leucocytes from certain monkeys release histamine following exposure to anti-human IgE. Both of these Rhesus leucocyte responses are potentiated by D2O. This potentiation of histamine release in vitro in two species by D2O was compared with the potentiation of cutaneous reactivity in IgE-mediated cutaneous reactions in two species. The addition of D2O to Ascaris antigen or anti-IgE increased the end-point cutaneous titres to these stimuli in Rhesus monkeys and the addition of D2O to Ascaris or ragweed antigen increased the end point cutaneous titre to these reactants in allergic dogs. D2O did not potentiate cutaneous reactivity to histamine in either dog or Rhesus monkey.     

The release of interleukin-1-like activity by macrophages in two models of immunological inflammation in the rat

Interleukin-1 (IL-1) is a putative mediator of inflammation released by activated macrophagesin vitro. The IL-1 release by rat macrophages collected either from exudates in pertussis-induced air pouches or from the peritoneum during adjuvant arthritis has been investigated. In air pouch inflammation LPS-stimulated macrophages collected from sensitized animals release more IL-1 than cells from control rats at day 6 after challenge. This enhanced IL-1 release parallels the extent of mononuclear cell migration in the inflammatory lesion. In adjuvant arthritis LPS-stimulated macrophages collected from sensitized animals release more IL-1 than cells from control rats at days 16 and 23 after adjuvant injection. The secondary inflammation in arthritic rats was statistically significant at days 16 to 28. These results indicate that during immunological inflammation macrophages either from the inflamed area or from a non-inflamed region release more IL-1 than control cells. This release parallels the extent of the inflammation and may be important in its pathogenesis.     

Occurrence of histamine-production-increasing factor in the postanaphylactic phase of allergic inflammation

Experimental study on histamine liberation in a postanaphylactic phase of allergy was carried out employing an air pouch model of allergic inflammation in rats. The antigen used was azobenzenearsonate-conjugated acetyl bovine serum albumin. Synthesis and liberation of histamine took place in the inflammatory pouch in the dorsum of the allergic rats, and brought about a gradual rise in the histamine level in the pouch fluid with a peak at 24 h after the antigen challenge. The time course of the histamine level in the pouch fluid was quite similar to that of histidine decarboxylase activities in the inflammatory tissues. alpha-Fluoromethylhistidine reduced the histamine level in the postanaphylactic phase, although it had been ineffective in the anaphylactic phase. A substance capable of increasing histamine production by bone marrow cells was found in the pouch fluid of allergic rats, while it was absent in the normal rat serum and pouch fluid of nonsensitized rats. The histamine-production-increasing activity rose until 24 h after the antigen challenge, but fell at 48 h in parallel with changes both of histamine levels in the pouch fluid and histidine decarboxylase activity in inflammatory tissues. The histamine-production-increasing factor is thought to be a protein, since it was inactivated by treatment with heat (70 degrees C for 30 min) or trypsin. Its molecular weight was estimated to be between 25,000 and 40,000.