Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggest that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.     

Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes.

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggests that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.     

Effect of L-tryptophan supplementation on eosinophils and eotaxin in guinea pigs

Eosinophilia Myalgia Syndrome is a hypereosinophilic disorder that appears to result from the ingestion of the dietary supplement L-tryptophan by susceptible individuals. It is unclear if this disease results from tryptophan, contaminants found in tryptophan, individual predisposition (such as immune status and allergies), or some combination of effects. To evaluate effects of L-tryptophan on eosinophil migration, guinea pigs were compared with or without supplemental tryptophan (0.4 g/kg/day), with or without immune sensitization, and with or without immune challenge. Eosinophil counts were obtained from bone marrow, blood, lung, and bronchial alveolar lavage fluid (BAL). Lung cells were obtained to measure eotaxin concentrations in supernates and lysates with or without antigen and calcium ionophore challenge using direct ELISA. Skin biopsies were taken from both non-injected and antigen injection sites. The tryptophan supplemented, antigen-sensitized/antigen-challenged guinea pigs showed a significant decrease in blood eosinophils, compared to control (cellulose) supplemented antigen-sensitized/antigen-challenged guinea pigs [(0.086 +/- 0.023) x 10(6) vs (0.147 +/- 0.021) x 10(6) eosinophils/ml recovered, respectively] with a significant increase in BAL eosinophils [(0.052 +/- 0.008) x 10(6) vs (0.033 +/- 0.005) x 10(6) eosinophils/ml recovered, respectively]. Unchallenged lung cell lysates from tryptophan-supplemented guinea pigs contained significantly less eotaxin compared to cellulose-supplemented guinea pigs regardless of whether they were sensitized (0.006 +/- 0.002 vs 0.027 +/- 0.008 ng/10(6) cells, respectively). No differences were observed in skin biopsies between cellulose and tryptophan groups. These results suggest that L-tryptophan-supplemented guinea pigs have altered eotaxin regulation, a potential mechanism by which human overconsumption of tryptophan dietary supplements could lead to hypereosinophilic disorders in susceptible individuals.     

Reactivity of rabbit antiserum to guinea pig eosinophils.

Although the eosinophil has been recognized as a distinctive cell type for almost 100 years, the major functions of these cells remain unknown. As an approach to defining these functions we have treated guinea pigs with rabbit antiserum to eosinophils (AES) in an attempt to ablate these cells from tissues. Rabbits were immunized thrice with purified eosinophils and the antisera were absorbed with peripheral blood cells from guinea pigs made eosinopenic with methyprednisolone to remove antibodies reactive with serum proteins and erythrocytes. The resulting sera reacted strongly with eosinophils in cytotoxicity tests and had weak or no reactivity with neutrophils. However, absorption of AES with purified neutrophils removed antieosinophil activity. Intraperitoneal injection of potent AES into guinea pigs resulted in complete absence of eosinophils from the peripheral blood and from the peritoneal cavity with only transient or no reduction in circulating neutrophils. Eosinophils were also reduced in bone marrow, spleen, and intestine. The ability of neutrophils to absorb AES activity in spite of weak reactivity in cytotoxicity tests may reflect a quantitative difference in antigenic determinants between eosinophils and neutrophils.     

The anti-allergic effects of Crinum glaucum aqueous extract.

The aqueous extract of Crinum glaucum was investigated for its effects on rat passive cutaneous anaphylactic reaction, rat peritoneal mast cell degranulation and allergic bronchoconstriction in the guinea pig. The extract demonstrated a significant (p < 0.05) reduction in area of dye leakage. The extract, administered for five days, inhibited mast cell degranulation of normal and passively sensitized rats induced by dextran and antigen. Allergic bronchoconstriction in actively sensitized guinea pigs was inhibited by the extract. The effects of the extract observed were comparable to those of sodium cromoglycate. These results substantiate the efficacy of the extract in the treatment of asthma, in traditional medicine.     

Legionella pneumophila intracellular growth in normal vs. Immune guinea pig macrophage cultures

Intracellular growth ofLegionella pneumophila, an opportunistic intracellular bacterium considered the cause of legionellosis, was assessed in peritoneal macrophages from normal and immunized guinea pigs. These bacteria grew exceedingly well in the normal guinea pig macrophages. Uptake of these bacteria was about the same by macrophages from either normal or immune guinea pigs, but their growth in immune macrophages was completely inhibited. Macrophages from normal guinea pigs stimulated with mezerin, a compound similar to diterpene ester, a known nonspecific stimulator of macrophages, or with specificLegionella vaccine released moderate or only small amounts of hydrogen peroxide, an indicator of macrophage activation to antimicrobicidal activity. In contrast, macrophages from immune guinea pigs produced much higher levels of hydrogen peroxide when stimulated with mezerein or theLegionella vaccine, and also showed a heightened response when cultured without a stimulator. These results indicate that macrophage activation related to the immune status of the host appears to have an important role in initial resistance toLegionella growth in susceptible individuals.     

RNA extracts of lymphoid cells sensitized to DNP-oligolysines convert nonresponder lymphoid cells to responder cells which release migration inhibition factor

Strain 13 nonresponder peritoneal exudate cells were converted to responder status to α or ?,DNP-oligolysines after incubation of the cells with RNA extracts prepared from responder guinea pigs skin test sensitive to these synthetic antigens. The conversion of nonresponder strain 13 cells was assessed by the direct cell migration inhibition correlate of delayed hypersensitivity. Nonresponder cells were not converted by RNA extracts prepared from unimmunized responder guinea pigs or from non-responder strain 13 guinea pigs previously injected with DNP-oligolysines. Thus, it seems possible to correct immunological unresponsiveness in vitro in spite of a specific genetically determined deficiency of the immune response related to the Ir gene.     

Transfer Agent of Immunity

An immune ribonucleic acid (RNA) preparation was extracted with phenol from the spleens of guinea pigs immunized with diphtheria toxoid. Antibody-carrying cells were detected by immunocyte adhesion as rosette-forming cells. When germ-free rats, conventional guinea pigs or mice were injected intraperitoneally with this preparation, the rosette-formers were detected in either peritoneal exudate cells or spleen cells, whereas serum antibodies were unable to be detected thus far in such animals. Two injections with this preparation did not cause any remarkable increase in the number of rosette-formers, and serum antibody was also not detectable. By contrast, a high titer of serum antibody was demonstrated and the number of rosette-formers increased shortly after an injection of a small amount of diphtheria toxoid into guinea pigs which had previously received an injection with immune RNA. This reaction indicates a secondary response of antibody formation. However, secondary responses were not induced by injections of immune RNA preparations in guinea pigs primed with either diphtheria toxoid or immune RNA preparation. These facts suggest that immune RNA preparations did not contain antigens or fragments thereof and the immune response induced by RNA preparation is not the same as that induced by stimulation by the antigen itself. These results moreover can be accounted for by the notion that the immune RNA preparation is able to induce “memory” cells capable of responding to a secondary stimulus with an antigen and producing a high titer of serum antibody.     

Langerhans cells: target cells in immune complex reactions.

Skin of normal, cobra venom extract-treated, and C₄-deficient guinea pigs was injected with ferritin-antiferritin or with peroxidase-antiperoxidase immune complexes. Skin and draining lymph nodes were studied to compare the phagocytosis of these immune complexes by Langerhans cells (LC) and by macrophages. When complement was present, immune complexes were damaging to LC, and uptake of the immune complexes, although present, was limited. When components of complement were absent or diminished, increased numbers of LC in lymph nodes were seen, but damage to LC was absent or decreased. However no detectable change in the amount of phagocytosis by LC was noted. Since some LC can carry antigen from skin to lymph nodes and may be involved in the presentation of antigen to lymphoid cells in some cell-mediated immune responses, impairment or abolition of LC function by immune complexes could represent a mechanism through which the local presence of antibodies might interfere with the induction and elicitation of cellular immunity by antigen. Moreover, damage to LC and subsequent release of intracellular (lysosomal?) substances may constitute a general mechanism of response in the skin to injury and may be an integral part of inflammatory and allergic skin reactions.     

Opsonizing activity of sera from animals immunized with pertussis vaccine

Opsonizing activity of guinea pig blood serum containing mercaptoethanol-resistant pertussis antibodies was studied in vitro on a model of microorganism ingestion by the mononuclears of the guinea pig peritoneal exudate. There were revealed distinct differences in the serum activity depending on the phagocytosis object. The blood serum of hyperimmunized rabbits stimulated the ingestion of Bordetella pertussis by mononuclears of guinea pigs--normal and immunized with pertussis vaccine. The blood sera of hyperimmunized guinea pigs and of mice immunized with pertussis vaccine twice displayed opsonins to B. pertussis. The blood sera of animals immunized with pertussis vaccine inhibited the staphylococcus ingestion by the peritoneal exudate mononuclears of guinea pigs, both normal and those immunized with pertussis vaccine.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

In vitro killing of S. mansoni schistosomula by eosinophils from infected rats: role of cytophilic antibodies.

The cytotoxic effect of peritoneal cells from Schistosoma mansoni-infected rats against antibody-opsonized or nonopsonized schistosomula in vitro has been studied during the course of infection. Eosinophil-enriched cell preparations were shown to have a high cytotoxic effect on schistosomula in the absence of antibody. The killer cells were identified as eosinophils. As in the ADCC mechanism previously described, mast cell-eosinophil interaction was required for eosinophil cytotoxicity. Rosette formation using S. mansoni antigen-coated erythrocytes was used to demonstrate the presence of anti-S. mansoni IgG2a antibody at the surface of infected eosinophils. Passive sensitization of normal eosinophils with ultracentrifugation pellets of immune rat serum resulted in a significant cytotoxicity of sensitized eosinophils. A close relationship was found between the cytotoxic activity of infected cells and the ability of the corresponding infected serum to arm normal eosinophils. At certain periods after infection, eosinophils from infected rats were less effective than normal eosinophils on antibody-coated schistosomula. EA- (rat) rosetting assay and blockade experiments with homologous immune complexes have revealed in a kinetic study that the blocking of cytotoxic activity of infected eosinophils was related to heat-stable circulating immune complexes. The possible role of immune complexes either in arming or inhibiting effector cells is suggested.     

Antibacterial activity of stimulated guinea pig peritoneal exudate cell culture supernates

Guinea pigs infected with Mycobacterium bovis BCG at least 5 weeks previously yield peritoneal exudate cells which, when stimulated in culture for 2–3 days with specific antigen (PPD), elaborate into the culture medium a factor which is antibacterial for listeria and corynebacteria, but not against other organisms tested. Concentrations of colchicine and vinblastine above 10^?7M completely inhibited its production. The yield of the factor was also found to be dependent on the choice of culture medium, the source of serum, and whether or not the scrum was heated at 56 °C for 30 min. It was weakly antagonized by the anions, RNA, and cyclic and noncyclic nucleotides, but not by lysozyme. It did not lyse M. lysodcikticus and had at least 100 times more activity than egg white lysozyme against listeria. Nonadherent cells from BCG-infected guinea pigs, plus PPD, stimulated adherent monolayer cells from normal guinea pigs to produce this material, but they did not stimulate adherent, cells from rabbit peritoneal exudates to its production.     

Characterization of in vitro reactivity by BCG-treated guinea pigs to syngeneic line-10 hepatocarcinoma

Summary The purpose of this study was to characterize in vitro the systemic tumor immunity induced by a BCG-intratumoral injection in line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2). Macrophages from BCG-tumor-cured guinea pigs at effector to target cell ratios of 10:1 and 100:1 were cytotoxic in vitro to line-10 tumor cells, and this cytotoxicity was potentiated by autologous serum. Significant cytotoxicity of lymphocytes from BCG-tumor-cured guinea pigs could only be achieved at ratios of 10,000:1, and no effect of autologous serum could be demonstrated. Lymphocytes from both normal and BCG-tumor-cured (line-10 immune) guinea pigs had a significant cytotoxic effect on the highly antigenic line-1 cells at ratios of 1:10,000. Macrophages from both normal and line-10 immune guinea pigs were cytotoxic to line-1 target cells at ratios of 1:100. With respect to specific cytotoxicity (cytotoxicity above and beyond levels achieved with effector cells from normal animals), the only significant difference was demonstrated when line-10 served as target cells and the effector cells were isolated from BCG-tumor-cured (line-10 immune) guinea pigs. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; CMEM, complete minimum essential medium; cpm, counts per minute; HBSS, Hanks' balanced salt solution; i.d. intradermally; i.p., intraperitoneally; PEC, peritoneal exudate cells; SDA, superficial distal axillary; ¹²⁵IdUrd, [¹²⁵I]iododeoxyuridine.     

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm ³H/mg tissue or as the percentage change in [³H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena.All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.     

Blocking of delayed hypersensitivity by humoral antibody in an in vivo mouse transfer assay using sensitized guinea pig cells

Humoral antibody was shown to interfere specifically with the expression of cell-mediated immunity (delayed hypersensitivity) in an in vivo system. Mice that received peritoneal exudate cells obtained from guinea pigs sensitized to 1-chloro-2,4dinitrobenzene (DNCB) exhibited delayed hypersensitivity reactions after challenge with the sensitizing agent. While control groups that received either normal sera, saline, or anti-BSA (bovine serum albumin) in addition to peritoneal exudate cells from sensitized guinea pigs exhibited positive delayed reactons to challenge with DNCB, mice that received anti-DNP (dinitrophenyl group) in addition to the senstized cells were prevented from exhibiting a delayed reaction to DNCB.     

Antigen presentation by liver sinusoidal lining cells after antigen exposure in vivo

The ability of liver sinusoidal lining cells (LSLC), a mixture of Kupffer cells and endothelial cells, to function as antigen-presenting cells (APC) was examined. Guinea pig LSLC were found to present antigen in vitro, albeit somewhat less effectively than a reference population of peritoneal exudate macrophages. The difference in APC function could not be explained by a deficiency of interleukin 1 (IL 1), as LSLC secreted IL 1 and expressed membrane-bound thymocyte stimulatory activity. The ability of LSLC to take up antigen from the portal blood in vivo and present it to primed T lymphocytes in vitro was also investigated. Trinitrophenyl-ovalbumin was injected intraportally into either strain 13 or strain 2 guinea pigs. The LSLC were subsequently isolated by collagenase digestion and density separation and assessed for the ability to induce proliferation of antigen-primed accessory cell-depleted syngeneic peritoneal exudate T lymphocytes in vitro. The in vivo antigen-pulsed LSLC were found to present antigen in vitro to primed T cells in an antigen-specific and genetically restricted manner. T cell DNA synthesis induced by antigen-bearing LSLC could be augmented by coculture with additional accessory cells, but not IL 1-containing macrophage supernatants. Enhancement of responsiveness was not genetically restricted. The demonstration that LSLC can take up, process, and retain antigen in vivo and present it to primed T cells in vitro suggests that LSLC are capable of contributing to the immune response to antigens appearing in portal blood.     

Ixodes dammini: Induced skin lesions in guinea pigs and rabbits compared to erythema chronicum migrans in patients with lyme arthritis

Erythematous skin lesions occurred in rabbits 2 days after being fed upon by larvae or nymphs of the tick, Ixodes dammini. Similar lesions occurred in guinea pigs 7 days after a primary infestation with either larvae or nymphs. Host resistance to secondary feeding by larvae was demonstrated in guinea pigs and rabbits. Host resistance to secondary feeding by nymphs was seen in guinea pigs, but not in rabbits. Guinea pigs developed resistance to nymphs after being previously fed upon twice by larvae. All skin lesions in resistant guinea pigs contained large accumulations of basophils (49–76% of cells) with smaller (20–33%), but significant, numbers of eosinophils. These responses were characteristic of strong cutaneous basophil hypersensitivity reactions. Primary and secondary lesions in rabbits fed upon by larvae contained mostly mononuclear cells (46–52%) and moderate numbers (16–30%) of basophils and eosinophils. Primary and secondary lesions in rabbits fed upon by nymphs had few (3–11%) basophils and eosinophils and were dominated by mononuclear cells (73–86%). Thus, acquired resistance in guinea pigs and rabbits was associated with cutaneous basophil and eosinophil responses and the lack of resistance of rabbits to nymphs was associated with erythematous lesions dominated by mononuclear cells. The mononuclear nature of rabbit lesions induced by nymphal feeding was similar to that seen in erythema chronicum migrans in Lyme arthritis patients who are thought to have been fed upon by I. dammini nymphs. This study confirms the cutaneous basophil hypersensitivity characteristics of lesions in guinea pigs resistant to ticks and demonstrates a relationship between the mononuclear cell response of rabbits to nymphal I. dammini and the cellular response seen in patients with erythema chronicum migrans and Lyme arthritis.     

Dose-response relationship in the migration inhibition test using peritoneal exudate cells and blood leucocytes of tuberculin sensitive guinea pigs

A comparison of the direct migration inhibition using peritoneal exudate cells and peripheral blood leucocytes from the same tuberculin sensitive guinea pigs was performed. Three antigen concentrations: 3, 15, and 75 microgram of PPD per ml were used. Both type of cells provided similar results except at early incubation intervals when leucocytes, in the presence of lower doses of the antigen, displayed stronger inhibition than peritoneal cells. Thus, in our study, peripheral blood leucocytes are at least equivalent to peritoneal exudate cells in the migration inhibition test.