Regulation of eosinophilia in rats infected with Nippostrongylus brasiliensis. I. Eosinophil chemotactic factor produced spontaneously by mesenteric lymph node cells of infected rats

The possible mechanism of eosinophilia was studied in rats undergoing primary infection with Nippostrongylus brasiliensis (Nb). In vivo studies showed that the kinetics of intestinal tissue eosinophilia was not directly related to those of the intestinal worm burden. Furthermore, Nb worm extract has no or only very weak in vitro eosinophil chemotactic activity, suggesting that parasite-derived eosinophil chemotactic factor (ECF) is, if at all, not a major regulator for intestinal tissue eosinophilia in this Nb rat system. On the other hand, when mesenteric lymph node (MLN) cells obtained various days after infection were cultured, potent ECF activity was detected in the cell-free supernatant from the cultures of MLN cells 15-20 days after infection, at which time marked intestinal tissue eosinophilia was observed in vivo. Production of ECF by MLN cells from Nb-infected rats seems to be spontaneous, since these cultures were performed without adding worm antigen. ECF-producing activity of day-20 MLN cells was suppressed by adding various metabolic inhibitors such as cycloheximide, mitomycin C, or puromycin. After Sephadex G-75 gel filtration, ECF activity produced by day-20 MLN cells was associated with two different molecules.     

Growth and differentiation in vitro of mast cells from mesenteric lymph nodes of Nippostrongylus brasiliensis-infected rats.

Intestinal mastocytosis begins to develop in rats, depending on the strain, at 14 (outbred Sprague-Dawley, SD) or 16 (inbred Lewis, L) days after infection with the nematode Nippostrongylus brasiliensis (Nippo). We have investigated in vitro mastopoiesis from mesenteric lymph node (MLN) cells cultured at various intervals post-infection, using a modified Marbrook liquid system. Greater increases in mast cells (MC) were observed in cultures of SD-MLN removed on day 14 after Nippo infection (IMLN-14) than from MLN removed from uninfected animals (NMLN): seven- to twenty-fold versus up to two-fold at 2 weeks and forty- to two hundred-fold versus up to twenty-fold at 4 weeks, respectively (P < 0.002). In contrast, similar differential increases in MC and histamine compared to uninfected controls, were demonstrated in 2 week cultures of MLN from L strain rats removed 17 (IMLN-17) and 20 (IMLN-20) but not 14 days after Nippo infection (P < 0.001). the presence of phytohaemagglutinin (PHA) in vitro was associated with enhanced MC differentiation from both IMLN-17 and IMLN-20, while worm antigen (Ag) stimulated mastopoiesis from IMLN-17, but suppressed the response from IMLN-20 (P < 0.02). Conditioned media (CM) prepared from unstimulated or PHA-stimulated IMLN-32 (i.e. removed 32 days after Nippo infection) caused significant mastopoiesis from NMLN in vitro when compared to no CM or Ag-stimulated CM (P < 0.01). Either MC precursors or cells which help MC differentiation exist in increased numbers in MLN of Nippo-infected rats. Mitogenic or antigenic stimulation modulates in vitro mastopoiesis, either directly or through soluble factors derivable from MLN cells. These in vitro methods can be utilized to understand further mechanisms of intestinal mastocytosis in the rat.     

Studies on in vitro IgE synthesis from lymph node cells of mice during infection with Nippostrongylus brasiliensis

The effect of serum factors on Ig synthesis (IgE, IgG) in vitro was analyzed. Spleen and mesenteric lymph node cells were obtained from Nippostrongylus brasiliensis-infected and non-infected mice. Sera and ammonium sulphate precipitated serum fractions from mice of different genetic origin (Balb/c - H-2^d, A.CA - H-2^f, B10.G - H-2^q) suppressed in vitro IgE synthesis whereas a pronounced enhancement of IgG antibody synthesis was obtained in several experiments. Our results obtained with sera from both high and low IgE responder strains demonstrated that no strain specificity exists as to the inhibitory efficacy of mouse sera for total IgE synthesis in vitro. The suppressive activity of the mouse sera was concentrated in a fraction precipitated with 20%–50% saturated ammonium sulphate. Amicon XM50 ultrafiltration suggested that this fraction had an apparent molecular weight >50,000 daltons. Suppressive activity was removed by immunoadsorption of the 20–50% fraction with anti-IgE Sepharose. After exogenous addition of monoclonal IgE to an inactive fraction in vitro neither the fraction enriched in IgE nor monoclonal IgE alone were able to suppress IgE synthesis in the culture. Our results suggest that one or more serum factors in the presence of IgE are responsible for the suppression of total IgE synthesis in vitro.Abbreviations BSA Bovine serum albumin - CFA Complete Freund's adjuvant - DNP Dinitrophenyl - EFA Enhancing factor of allergy - Fc R Fc-receptor for IgE - FCS Fetal calf serum - MLN Mesenteric lymph node - PBS Phosphate-buffered saline - PHA Phytohemagglutinin - PPD Purified protein derivative - SFA Suppressive factor of allergy     

IgE antibody production is associated with suppressed interferon-gamma levels in mesenteric lymph nodes of rats infected with the nematode Nippostrongylus brasiliensis.

IgE and IgG2a antibody production and interferon (IFN)-gamma secretion were studied in rats infected with the gut nematode Nippostrongylus brasiliensis by in vitro cultivation of mononuclear cells obtained from spleen (SPL), mesenteric lymph nodes (MLN) and pulmonary hilar lymph nodes (PLN). The highest levels of IgE were detected in the culture supernatants of MLN cells after infection: IgE levels were modest in PLN and negligible in SPL. In contrast, the highest levels of IgG2a were produced by PLN cells, followed by MLN and SPL cells. These results indicate that the MLN is the most significant site for IgE production in nematode infection, while IgG2a production is more marked in PLN. In naive rats, the spontaneous secretion of IFN-gamma was highest in PLN cells, followed by MLN and SPL cells. After the infection, IFN-gamma levels were significantly decreased in MLN and PLN. Suppression of IFN-gamma secretion was also observed in concanavalin A (ConA)-stimulated MLN and PLN cells from infected rats. In MLN, the ratio of CD4+ to CD8+ T cells was increased after the infection. Stimulation with an allergen-rich, excretory-secretory (ES) substance of the nematode enhanced ongoing IgE production, and suppressed IFN-gamma secretion by MLN and PLN cells. In contrast, an allergen-poor, adult worm extract potentiated IFN-gamma secretion. These results show that nematode-induced IgE antibody response is associated with the suppressed production and/or secretion of IFN-gamma, particularly in the MLN, and that some molecules in the ES substance may trigger these immune responses.     

Mast cell responses in mesenteric lymph nodes to infection of rats with the nematode, Nippostrongylus brasiliensis

The number of mast cells and their distribution in rat mesentery lymph nodes were assessed after a primary infection and after several successive infections with the nematode, Nippostrongylus brasiliensis. Following primary infection with N. brasiliensis, two peaks in total mast cell counts were observed. An initial small increase was restricted to day 5 and to the region of entrance to the lymph node. During the second peak, a marked increase in the number of mast cells occurred after day 15, the majority of cells is migrating through the afferent lymphatics, and then advancing from the cortical to the medullary region. The number of cells found in the hilus always remained low, indicating that mast cells accumulate and degranulate within the lymphoid organ.

In rats infected several times with the nematode parasite, mast cell numbers were markedly increased and the distribution pattern was similar to that found on day 21 after a primary infection. The observation that the percentage of cells found in the capsule was rather low in these animals indicates that local proliferation might have contributed to the high mast cell counts.

     

Reagin-like antibodies in rats infected with the nematode parasite Nippostrongylus brasiliensis

The immune response to Nippostrongylus brasiliensis in rats is closely associated with the formation of antibodies resembling human reagins. These are detected in some rats immediately after acquiring resistance to the parasite after an initial infection and in all rats 1 week later. Further infections stimulate an anamnestic rise in `reagin' production. `Reagin' formation is due to the living worms, primarily the adult stage. `Reagins' were detected by passive cutaneous anaphylaxis in the rat (homologous passive cutaneous anaphylaxis=PCA) with a 72-hour interval between intradermal injection of antibody and intravenous injection of antigen, which was a saline extract of adult worms.

`Reagin' production in rats vaccinated with worm extracts contrasts sharply with `reagin' production in rats infected with living worms. Vaccination with worm extracts stimulates `reagin' production in only some rats during the primary response only; after second and later vaccinations, `reagins' were not detected in any rats. Prolonged vaccination induces the formation of `blocking' antibodies and a further type of antibody capable of inducing 6-hour PCA, but not 72-hour PCA in rats. Even after prolonged vaccination, however, a subsequent infection stimulates an anamnestic rise in `reagin' production in some rats. Vaccination did not induce resistance to infection in any rats.

Rats recovered from an initial infection become increasingly susceptible to anaphylactic shock when challenged with worm antigen, but the sensitivity of rats to systemic anaphylaxis is not closely correlated to the level of circulating `reagins'. Heterologous anaphylactic reactions did not induce expulsion of worms from the intestine and anaphylaxis is not directly related to the ability of rats to resist infection.

     

Mast cell differentiation in cultures of T cell-depleted mesenteric lymph node cells from Nippostrongylus brasiliensis-infected mice

Lymphoid and bone marrow cells from normal and horse serum-immunized mice and lymphoid cells from Nippostrongylus brasiliensis-infected mice were cultured on monolayers of embryonic skin fibroblasts to analyze the factors which regulate the differentiation and proliferation of mast cells in vitro. Our results indicate that T cells can regulate the development of mast cells in vitro by either enhancement or suppression. In cultures of horse serum-immune spleen cells, inducer T cells are required for mast cells to develop. However, in cultures of mesenteric lymph node cells from N. brasiliensis-infected mice, inducer T cells are not required for mast cell development. This suggests that the development of mast cells may occur at discrete interleukin-3 (IL-3)-dependent and IL-3-independent stages. Mast cell precursors in the mesenteric lymph nodes of N. brasiliensis-infected mice may have already been acted on by inducer cells in vivo to become mast cell committed. While IL-3 does not appear to be required for mast cell development, these precursors do require the presence of a connective tissue microenvironment such as embryonic skin. The precursors can be inhibited from development by interferon preparations.     

Studies on the mechanism of the self-cure reaction in rats infected with Nippostrongylus brasiliensis

The production of ovalbumin-induced anaphylaxis in rats passively immunized with antiserum to Nippostrongylus brasiliensis produced a significant reduction in their transplanted populations of adult worms compared to: (a) rats which were passively immunized alone, and (b) rats subjected to ovalbumin anaphylaxis alone.

This result suggests that the physical changes associated with anaphylaxis facilitated the passage of antibody into the sub-epithelial spaces and intestinal lumen where its effect was specifically directed against the worms.

Since in naturally infected rats an intestinal lesion is present, which grossly and microscopically resembles that induced by ovalbumin anaphylaxis, it was concluded that this lesion plays a similar role in the self-cure reaction.

     

Adoptive transfer of total and parasite-specific IgE responses in rats infected with Nippostrongylus brasiliensis.

Infection of rats with Nippostrongylus brasiliensis has both a parasite-specific and non-specific IgE stimulating effect. Both these responses can be adoptively transferred with thoracic duct lymphocytes (TDL) from infected rats. The character of the IgE response in the recipient rats was related to the stage after infection of the cell donors. TDL from hyperimmune rats adoptively transferred high serum titres of parasite-specific IgE to infected recipient rats and substantially increased the levels of total IgE. However, adoptive immunization with TDL from donors infected 10 days previously did not stimulate parasite-specific IgE and only slightly increased total IgE levels. After cell fractionation the sIg- cells from day 10 TDL increased the level of total IgE but not parasite-specific IgE whereas sIg- cells from hyperimmune TDL did not induce any IgE response unless given with sIg+ cells. The possible reasons for this are discussed.     

The production of IgE and IgGa antibodies in normal rats and rats infected with Nippostrongylus brasiliensis.

The time courses of production of IgE and IgGa homocytotropic antibodies were measured in Wistar rats during a primary and secondary response to egg albumin with pertussis or Freund's adjuvants. An anamnestic IgE antibody response occurred in animals previously sensitized to antigen with killed Bordetella pertussis as adjuvant. IgGa antibodies were formed in the primary response with Freund's complete adjuvant only, but were found during the secondary response with all adjuvants used. The time courses of formation of IgE and IgGa antibodies were very different during the secondary response. The production of both classes of antibody to egg albumin was studed in Wistar and Hooded Lister rats infected with Nippostrongylus brasiliensis. IgGa antibody formation was not potentiated by the infection. However, increased levels of IgE antibody, formed during a secondary response to antigen in infected animals, were consistently higher in both strains than during a primary response.     

IL-4 production by T depleted cells from Nippostrongylus brasiliensis infected mice

Interleukin-4 (IL-4) is known to be involved in both the in vivo IgE response and the elevated B cell IgE Fc receptor (Fc epsilon R11) expression seen after a parasite infection. To further analyze the relationship between Fc epsilon R11 expression and IL-4 production, purified B cells from uninfected, Nippostrongylus brasiliensis (Nbr) infected and from goat anti-mouse IgD (GaM delta) injected mice were isolated on various days post-treatment. The Fc episolon R11 levels on purified B cells from normal mice decreased after an overnight culture in media alone and addition of IL-4 to these cultures resulted in a 4 to 13-fold enhancement of Fc epsilon R11 levels. In contrast, the Fc epsilon R11 levels on B cells from Nbr infected mice were elevated after an overnight culture in media alone and addition of IL-4 did not further enhance the already upregulated Fc epsilon R11 levels. Overnight culture of purified B cell blasts from Nbr infected mice in the presence of an anti-IL-4 monoclonal antibody (11B11) caused the elevated Fc epsilon R11 levels to return to levels seen in normal mice, without affecting the Fc epsilon R11 levels on purified Go or B cell blasts from uninfected mice or Go B cells from Nbr infected mice. 11B11 also inhibited the elevated Fc epsilon R11 levels on highly purified B cells obtained by FACS sorting the non-adherent spleen cell population for class II+ cells. In contrast to Nbr infection, the Fc epsilon R11 levels on B cells were downregulated in the GaM delta injected mice. However, analogous to the Nbr system, the Fc epsilon R11 levels were unresponsive to the addition of exogenous IL-4. This study indicates that IL-4 production is seen in T depleted splenocytes and that this alternate source of IL-4 serves to maintain the elevated Fc epsilon R11 levels on B cells.     

Characteristics of histamine secretion from rat peritoneal mast cells sensitized to the nematode Nippostrongylus brasiliensis

Peritoneal mast cells from rats immunized with the nematode Nippostrongylus brasiliensis released histamine on challenge with antigen in both the presence and absence of added calcium. The response under the latter conditions was abolished by depletion of sequestered stores of the cation and probably reflected mobilization of these stores. The release was potentiated by brief pretreatment with chelating agents and inhibited (in the absence of phosphatidyl serine, PS) by supramaximal concentrations of calcium. Calcium bound to superficial sites in the membrane may then regulate the secretory process. Histamine release in both the presence and absence of calcium was enhanced by adenosine. Only the former response was potentiated by PS, in keeping with the view that the lipid promotes influx of the cation from the extracellular environment. The activated state induced by antigen decayed with time but PS slowed this process. Histamine secretion in the absence of calcium was more rapid than in the presence of the cation, consistent with the release of bound calcium in a pulse of short duration.     

Mast cells and macromolecular leak in intestinal immunological reactions. The influence of sex of rats infected with Nippostrongylus brasiliensis

The onset of the exponential expulsion of Nippostrongylus brasiliensis worms in the rat is associated with a sharp burst of intestinal mast cell activity and increased permeability of the bowel wall. It was found that the onset of maximum velocity of worm expulsion occurred earlier in female rats than in male rats and proceeded at a faster rate. There was a corresponding difference in the timing of the mast cell rise and macromolecular leak between the sexes. This suggested that there is a relationship between these events. Cortisone, a drug known to stop worm expulsion and to suppress the mast cell response, also prevented the macromolecular leak. Electron microscopy showed that during the period of increased permeability a pathway for protein tracers had opened up between the epithelial cells and it is suggested that this is the route for enhanced antibody transfer across mucous membranes.We suggest that a stimulus or stimuli from the parasite cause synchronous development of new populations of mast cells, IgE-producing plasma cells and plasma cells synthesizing antibodies of other classes possessing an anti-worm effect. It is also suggested that these mast cells discharge their pharmacological mediators by an allergen—reaginic antibody mediated system and that these mediators create a pathway through the intestinal mucosa for the translocation of antibody.     

Chymase inhibitor ameliorates eosinophilia in mice infected with Nippostrongylus brasiliensis

BACKGROUND: Chymase is a chymotrypsin-like serine protease primarily stored in mast cells. Infection with helminth parasites is known to increase the level of mast cell chymase in the jejunum and serum in mice. The aim of the present study is to elucidate the role of chymase in helminth infection. METHODS: Chymase inhibitor SUN-C8257 was administered to mice infected with the nematode Nippostrongylus brasiliensis, and the number of eosinophils in the blood, serum IgE levels and fecal egg counts were determined. RESULTS: Administration of SUN-C8257 significantly inhibited blood eosinophilia in BALB/c mice infected with N. brasiliensis. The effect of SUN-C8257 was specific for eosinophils, in that it affected neither the number of total leukocytes nor serum IgE levels. SUN-C8257 did not alter the fecal egg counts in this model, showing that SUN-C8257 has no effect on infectivity and expulsion of the nematode. N. brasiliensis infection induced eosinophilia in mast cell-deficient mice (W/W(v)) as well as their littermates (+/+), and SUN-C8257 inhibited the eosinophilia in +/+ mice but not in W/W(v) mice. These results suggest that the eosinophil number may be regulated by different mechanisms in W/W(v) and +/+ mice, and that the effect of SUN-C8257 on nematode-induced eosinophilia is probably due to chymase inhibition. CONCLUSIONS: Chymase released by activated mast cells may play a role in helminth-induced eosinophilia.     

Origins of serum IgE and of the homocytotropic antibody-secreting cells in the thoracic duct lymph of rats infested with Nippostrongylus brasiliensis

Mesenteric lymphadenectomy caused a marked reduction in the amounts of both parasite-specific and total IgE in the serum of Nippostrongylus brasiliensis infested rats. This was accompanied by reduced quantities of IgE in the thoracic duct lymph, showing that the mesenteric lymph node is of great importance in the generation of the elevated IgE levels which accompany this infestation. Conversely, the intestinal lamina propria and the Peyer's patches did not appear to be significant contributors to either thoracic duct lymph or serum IgE levels. Homocytotropic antibody-secreting cells specific for parasite antigens were detected in the thoracic duct lymph of lymphadenectomized rats and are believed to arise directly from the gut mucosa and possibly from Peyer's patches.     

Sensitivity of passive cutaneous anaphylaxis in rats. II. Suppression of passive cutaneous anaphylactic reactions in rats infected with Nippostrongylus brasiliensis

The sensitivity of passive cutaneous anaphylactic (PCA) reaction was examined in rats infected with the nematode parasite, Nippostrongylus brasiliensis (Nb). PCA reactions with anti-DNP IgE antibody were remarkably suppressed by Nb infection already 10 days after infection and persisted for at least 28 weeks. 10 days after infection, no anti-Nb IgE antibody was present in the sera of infected rats, whereas 18 or 28 weeks after infection, circulating anti-Nb IgE antibody was present. It was concluded that two different mechanisms might explain the suppression. 10 days after infection, nonspecific IgE induced by potentiated IgE production inhibited passive sensitization with anti-DNP IgE antibody by occupying the IgE receptors on mast cells, whereas 18 or 28 weeks after infection, the mast cell IgE receptors were occupied by IgE antibody specific for the Nb antigen. The reactivity through non-immunological processes (skin sensitivity to compound 48/80) was not modified by Nb infection at any time after infection.