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.     

STUDIES IN EXPERIMENTAL EOSINOPHILIA : VI. Uptake of Immune Complexes by Eosinophils

A method is described whereby immune complexes may be visualized in a single cell. Bovine serum albumin labeled with a red-fluorescing dye was joined to a rabbit antiserum labeled with a green-fluorescing dye to yield an immune complex which fluoresced yellow when illuminated by ultraviolet light. Such yellow-fluorescing immune complexes were injected into the peritoneal cavity of guinea pigs, and the peritoneal exudates were examined subsequently. Yellow fluorescent particles were seen in eosinophils obtained from guinea pigs sensitized to hemocyanin and from normal animals. Eosinophils of the blood and of the bone marrow could also take up the complexes in vitro. Neither antigen nor antibody alone was taken up by eosinophils, nor was a mixture of labeled antigen and labeled normal globulin. Similar observations were made with human blood eosinophils. These experiments suggest that eosinophils act as part of the defense against the pathogenic effects of certain immune complexes.     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

Isolation of guinea pig macrophages bearing surface C4 by fluorescence-activated cell sorting: correlation between surface C4 antigen and C4 protein secretion

Studies of complement (C) secretion by single cells indicate that only a subset of a guinea pig macrophage population is capable of secreting the second (C2) and fourth (C4) components of C. Moreover, cell-surface bound C4 antigen is also found on a proportion of guinea pig peritoneal macrophages. The availability of methods for the isolation of macrophages bearing surface membrane C4 antigen and for the detection of the secretion of C by single cells made it possible to ascertain the relationship between these two subsets. Approximately 25% of the freshly isolated peritoneal macrophages were surface C4 antigen positive. When incubated in conditioned medium containing C4 or incubated in small volumes to increase the concentration of fluid phase C4, the proportion of macrophages bearing surface C4 increased to approximately 80%. The surface C4 antigen was adsorbed from the medium and was predominantly native C4. The proportion of peritoneal macrophages secreting functionally active C4 or C2 was approximately 45% as measured by a hemolytic plaque assay technique. The proportion of C4-secreting cells decreased to 5% after incubation in conditioned medium containing preformed C4, whereas the proportion of C2-producing macrophages was unchanged, i.e., it remained at about 45%. The removal of secreted C4 with F(ab')2 anti-C4 or effectively decreasing C4 concentration by increasing the volume of the culture medium abrogated the decrease in the proportion of C4-secreting cells. Conditioned medium derived from cells genetically deficient in C4 had no effect on the proportion of C4- or C2-producing macrophages. The macrophage population bearing surface membrane C4, isolated by fluorescence-activated cell sorting, contained more than 90% of the C4-producing cells. Cells producing C2 were distributed equally in both subpopulations. Maintenance of the surface C4-positive, C4-producing cells in culture for 12 hr resulted in a decrease in the proportion of C4-secreting cells. Conversely, isolated macrophages initially surface C4 negative and not producing C4, developed the capacity to produce C4 in culture. C4 production in the isolated surface C4-negative population was inhibited by incubation in medium containing preformed C4. These results suggest the presence of a negative feedback effect on C4 secretion that is mediated by extracellular C4.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro studies with "transfer factor". II. Transfer of the cell-migration inhibition correlate of delayed hypersensitivity in humans with nondialyzable components of leucocyte lysates from humans sensitized to histoplasmin and coccidioidin

“Nonsensitive” cells from skin-test-negative individuals were incubated with whole cell lysates, nondialyzable and dialyzable fractions of leucocyte lysates obtained from strongly skin test sensitive histoplasmin, and/or coccidioidin sensitive donors and specific antigen. “Nonsensitive” cells incubated with whole cell lysate or nondialyzable fractions of lysate in the presence of specific antigen released a substance (MIF) which specifically inhibited the migration of guinea pig macrophages. Dialyzable fractions from active cell lysates incubated with “nonsensitive” cells and specific antigen did not liberate MIF from “nonsensitive” cells. Cell lysates, nondialyzable fractions, or dialyzable fractions alone, or in combination with nonspecific antigens in the presence of “nonsensitive” cells failed to inhibit the migration of guinea pig macrophages.     

Detection of guinea pig macrophages by a new CD68 monoclonal antibody, PM-1K

A new monoclonal antibody, PM-1K, was raised against 24-h cultured human peritoneal macrophages. In immunohistochemical assays, PM-1K recognized freshly isolated blood monocytes and most tissue macrophages as well as myeloid dendritic cells such as Langerhans cells and interdigitating cells. The molecular size of the antigen recognized by PM-1K was determined to be 110 kD by means of immunoaffinity purification. Because this affinity-purified antigen recognized by PM-1K was also recognized by anti-CD68 antibodies, it is believed to be one of the heterogeneous molecules of the CD68 antigen. Analysis showed interspecies reactivity of PM-1K with macrophages from guinea pigs, pigs, bovine species, and monkeys. Among these macrophages, those of the guinea pig reacted strongly with PM-1K. Patterns of PM-1K immunostaining in guinea pig tissues were similar to those found in human tissues. Studies with the immunoelectron microscope revealed reaction products of PM-1K in the cytoplasm, especially around endosomes. Since only a few antibodies are available to label guinea pig macrophages, PM-1K is considered to be one of the most suitable antibodies to examine macrophages in experimental guinea pig models.     

Disappearance of macrophage surface folds after antibody-dependent phagocytosis

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon- aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature- dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer- 1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF is discussed.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Inhibitory effects of various synthetic substrates for aminopeptidases on phagocytosis of immune complexes by macrophages

The inhibitory effects of various 7-(aminoacyl)-4-methylcoumarinylamides (AA-MCA's), synthetic substrates for aminopeptidases, on phagocytosis of immune complexes by guinea pig peritoneal macrophages were investigated by measuring the intracellular uptake of sensitized 51Cr-sheep erythrocytes and 125I-alpha-amylase complexed with homologous IgG2 antibody. Among the AA-MCA's examined, MCA compounds of hydrophobic amino acids (Phe, Tyr, Leu, and Pro) were found to inhibit the intracellular uptake and digestion of immune complexes. Sucrose density gradient centrifugation of the homogenates of macrophages treated with the inhibitors for 1 h at 37 degrees C showed that they modulated the lysosomes, resulting in a decrease in buoyant density of the organella. These effects of the inhibitors on the buoyant density of the lysosomes as well as on the phagocytic activity of macrophages disappeared upon removal of the inhibitors from the cells by washing. Since none of 7-amino-4-methylcoumarin, L-phenylalanine, and bestatin methyl ester could significantly inhibit the phagocytosis of immune complexes by macrophages, the MCA compounds of hydrophobic amino acids appear to inhibit the phagocytosis as a consequence of their lysosomotropic nature.     

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen

Summary As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.Abbreviations ABA azobenzenearsonate - BSA bovine serum albumin - CFA complete Freund's adjuvant - IFA incomplete Freund's adjuvant - KLH keyhole limpet hemocyanin - LNC Lymph node cells - MHC major histocompatibility complex - PEC peritoneal exudate cells - PEL peritoneal exudate lymphocytes - RAN 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate - RAT L-tyrosine-azobenzene-p-arsonate - TAT L-tyrosine-azobenzene-p-trimethylammonium chloride Aided by USPHS Grant AI 05664.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. III. Long-term C4 suppression is actively maintained by a soluble suppressor factor (FsC4)

Previous work in our laboratory established that individual complement components can be regulated in vivo by administration of specific antibody or immunocompetent cells to newborns and in vitro by administration of specific antibody to cultured peritoneal macrophages or splenic fragments. Antibody-induced suppression of C4 was much longer lasting in cultured guinea pig splenic fragments than in cultured guinea pig peritoneal macrophages, suggesting that splenic fragments contained elements necessary for long-term suppression that were not present in the macrophage monolayers. This publication presents data in support of this concept. Antibody-treated splenic fragments from normal guinea pigs--but not from C4-deficient guinea pigs--elaborated a soluble factor (FsC4) that suppressed C4 production in previously untreated splenic fragments. FsC4 activity was most potent in splenic fragment culture supernatants at those times when intracellular and secreted C4 hemolytic activity and C4 antigen were at their lowest. C4 itself or a fragment of C4 was therefore unlikely to mediate suppression in this system. Residual anti-C4 antibody was ruled out as a mediator of FsC4 activity since it was shown by two independent methods that the amount of anti-C4 antibody carried over with the supernatant was orders of magnitude less than the amount necessary to cause suppression or to neutralize fluid phase C4 in fresh splenic fragment cultures. Preliminary data revealed that FsC4 activity may be mediated by two or more distinct molecular species or may be mediated by a single molecule that exhibits secondary size and charge heterogeneity. The identification of factors that are capable of regulating C4 suggests that, as with immunoglobulins, complement components may be regulated by complex networks of immunocompetent cells and their soluble products.     

Accessory cell function of cells of the mononuclear phagocyte system isolated from mycobacterial granulomas

Epithelioid cells from BCG-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas were examined for their ability to act as accessory cells for T-cell proliferation to mitogen (Con A) and antigen (PPD). The granuloma cells were separated on a FACS using monoclonal antibody specific to guinea pig macrophages. Epithelioid cells (which are Ia negative) were able to support proliferation to Con A but not to antigen. Cultures containing Ia positive granuloma macrophages from M. leprae sensitized animals did not show responsiveness to Con A or to PPD. Oil-induced peritoneal exudate macrophages from BCG or M. leprae immunized animals were able to act as accessory cells for both mitogen and antigen proliferation. The nonresponsiveness of cultures containing epithelioid cells stimulated with PPD or M. leprae granuloma macrophages stimulated with Con A was not due to suboptimal or supraoptimal accessory cell:lymphocyte ratios.     

Nature of T lymphocyte recognition of macrophage-associated antigens. IV. Inhibition of peptide antigen presentation by brief treatment of macrophages with anti-Ia serum

To examine the role of macrophage la antigens in T-lymphocyte stimulation, guinea pig macrophages were briefly treated with anti-Ia serum before or after antigen pulsing with the peptide antigen human fibrinopeptide B (hFPB). To assess their antigen-specific stimulatory capacity, the variously treated macrophages were added to culture with hFPB-immune guinea pig T cells and stimulation was determined by the incorporation of [³H]thymidine. Macrophages treated with anti-Ia serum before antigen pulsing stimulated T-cell responses equivalent to those observed with antigen-pulsed macrophages treated with normal serum. By contrast, brief anti-Ia treatment of macrophages immediately following a 2-hr antigen pulse reduced subsequent T-cell responses by 45 to 70%. Similar treatment of macrophages pulsed with antigen for only 1 hr produced only modest inhibition of T-cell responses. However, if macrophages pulsed for 1 hr with antigen were incubated several hours before brief anti-Ia serum treatment, the subsequent T-cell responses were reduced by 40 to 60%. This inhibition was specific for antiserum directed against Ia antigens of the guinea pig MHC, since brief macrophage treatment with antiserum directed against B.1 antigens, the guinea pig equivalent of murine H-2K and H-2D antigens, produced no inhibition of their T-cell stimulatory capacity. These results are discussed with respect to the formation of the immunogen presented by macrophages for T-cell recognition.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

A reevaluation of rabbit anti-allotype antibody for the presence of cross-reactive idiotypes. I. A species-specific idiotype on rabbit anti-a1 antibody is recognized by guinea pig anti-IdX antibody

Rabbit reagents previously thought to display specificity for a cross-reactive idiotype on anti-VHa allotype antibody from all tested rabbits have recently been shown to be contaminated with an induced (latent) molecule similar or identical to the original antigen (rabbit a1 or a2 allotype). In an attempt to circumvent this problem, we have immunized guinea pigs with rabbit anti-a1 allotype antibody to produce heterologous anti-idiotype antibody. The resulting guinea pig antibody (GP anti-R IdX) recognizes anti-a1 antibody from each of 17 immunized rabbits, and in four tested samples reacts with 22 to 100% of the molecules. Neither goat nor guinea pig anti-a1 reacts with the guinea pig anti-R IdX antibody, even though the goat, guinea pig, and rabbit anti-a1 Ab all recognize a similar set of a1 determinants. The reaction between IdX-bearing rabbit anti-a1 and guinea pig anti-R IdX is inhibited by the original antigen (a1 IgG), demonstrating that the IdX is at or near the antigen combining site of anti-a1 antibody. Immunoelectron microscopy of immune complexes supports this conclusion and demonstrates that the reactive site on the GP anti-R IdX is at or near its antigen combining site.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.     

Identification of the principal cell type yielding immune RNA capable of transferring tumor-specific cellular cytotoxicity

A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC.