Circulating immune complexes and in vitro cell reactivity in paracoccidioidomycosis

The severest forms of paracoccidioidomycosis (Pcm) are associated with impaired cell-mediated immunity, a phenomenon that is reversible with therapy. It has been postulated that plasma factors could be responsible for such immune dysfunction. In this report, circulating immune complexes (CIC) were measured by the Raji cell radioimmunoassay (Raji) and by the¹²⁵I-C1q binding assay (C1q-BA) in sera from 14 patients with either active or inactive forms of Pcm and from 15 healthy controls. The C1q-B A revealed significantly elevated levels of CIC in the sera of all but one of the patients. Four of the 8 active (62%) and 2 of the 6 inactive (33%) patients had CIC levels significantly higher than the controls as determined by the Raji test. Significantly increased levels of CIC were detected only in the active patients by the Raji test. The serum of one of the patients, with a generalized infection and depressed lymphocyte responsiveness, was examined and found to contain a factor which depressed the in vitro proliferation of both homologous and normal lymphocytes. We also found that pre-culture of the patients' lymphocytes before stimulation restored their proliferative capacity, and IC were detectable in the culture supernatants. However, the subsequent addition of the patients' serum to such precultured cells did not reinduce the depression. It is suggested therefore, that the depression of T cell responses observed in Pcm is due to the presence of IC which may interact reversibly with the responding cells and/or activate a suppressor cell population whose activity is diminished by preculture.     

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.     

Neoantigen of the complement membrane attack complex of cytotoxic human peripheral blood lymphocytes.

Neoantigenic determinants (neoAg) specific for the assembling membrane attack complex (MAC) of complement were detected by immunofluorescence microscopy on the surface of cytotoxic lymphocytes during the antibody-dependent cellular cytotoxicity (ADCC) reaction. This study employed antibody-sensitized chicken erythrocytes as target cells, human peripheral blood lymphocytes as effector cells, and RITC-conjugated rabbit F(ab')2-anti-neoAg. NeoAg was present on 60% of ADCC plaque-forming lymphocytes (PFL). Eight out of 182 neoAg-positive PFL were observed in direct contact with their target cells. In these cases MAC-specific neoAg was visualized at the zone of contact between the cells. Anti-neoAg Ig was found to inhibit ADCC plaque assays up to 62%; and 51Cr-release assays up to 79%. Stimulation of lymphocytes by PHA or mixed lymphocyte culture increased the expression of neoAg. In the case of PHA, increased neoAg expression was correlated with an increased incorporation of 14C-leucine into C5, C6, C7, and C8 antigens, which was detected by immunodiffusion and autoradiography.     

Cell-serum factor interaction. Characteristics of stimulation of protein synthesis in Ehrlich ascites cells by factors in serum and in cell extracts.

Stimulation of protein synthesis induced by serum in serum-starved Ehrlich ascites tumor cells was directly proportional to the concentration of added serum, inversely proportional to the concentration of cells, in the culture, and dependent on the length of exposure of cells to serum. Stimulation was markedly decreased in cells incubated with serum at temperatures lower than 37 °C. During the exposure of cells to serum, active protein synthesis was not required in order for subsequent stimulation of protein synthesis to take place. These characteristics were consistent with the possibility that stimulation of protein synthesis followed uptake of serum factors by cells. Extracts of cells stimulated protein synthesis in a similar fashion to serum. Stimulations by extracts and by serum were additive. The factors in cell extracts were macromolecular, associated with articulate fractions, and inactivated by trypsin, but not by RNAase, DNAase, ether or chloroform. Extracts of serum-grown cells were more stimulatory than extracts of serum-starved cells. When serum-starved cells were incubated with serum, stimulatory activities of their extracts increased as a function of time of incubation with serum.     

A suppressor cell which interferes with antiallotype stimulated DNA synthesis of rabbit B cells

Responsiveness of rabbit spleen cells to anti-allotype antibody was measured in terms of increased thymidine incorporation. Incorporation was enhanced after removal of cells which had ingested or had adhered to magnetic particles. B lymphocytes, prepared from spleen cells by the removal of adherent cells and of RTLA bearing T cells, were more responsive to anti-allotype antibody than were the original spleen suspensions. This increase could not be explained by enrichment in B cells. It was concluded that an adherent cell suppressed B cell transformation. The addition of 2-mercaptoethanol to the cell cultures stimulated with mitogen augmented the incorporation of thymidine. Adherent cells interfered with 2-mercaptoethanol potentiation in the response to anti-allotype antibody but not in the response to Con A. Fractionation of spleen cells, over glass bead columns, yielded nonadherent and adherent cell populations. The responsiveness of nonadherent cells to anti-allotype induced thymidine incorporation was two to six times that of unfractionated cells. The responsiveness of nonadherent cells to stimulation by anti-allotype antibody was reduced after addition of adherent cells. Findings were discussed in terms of the inhibitory role played by adherent cells on anti-allotype antibody induced responsiveness of rabbit B cells and of the possible participation of a third cell type which functions as a promotor of mitogenic T cell stimulation.     

Lymphocytes stimulated by allogeneic B cell lines cleave the third component of complement and fix C3 fragments. Their nonspecific lytic capacity is elevated against complement receptor type 2-carrying targets

Human blood lymphocytes stimulated in mixed cultures by allogeneic B cell lines were shown to cleave C3 molecules. The B cell lines were derived from Burkitt lymphoma patients: 1) established from their EBV-negative lymphoma, 2) the EBV-positive sublines converted in vitro, and 3) lymphoblastoid cell lines (LCL) i.e., B lymphocytes transformed in vitro by EBV. These cell lines differed considerably in their capacity to stimulate allogeneic lymphocytes. The split products of C3 were detected in the supernatants and on the surface of the activated lymphocytes at levels which correlated with the strength of stimulation. Lymphocytes cultured with LCL had the highest levels of thymidine incorporation blast transformation, C3 cleavage, and C3 fragment fixation. Lymphocytes exposed to the EBV-negative Burkitt lymphomas were stimulated weakly and their C3-activating capacity was low. Irrespective of the efficiency of lymphocyte stimulation induced in the cultures, 60 to 70% of the blasts were found to fix C3 fragments. The majority of the lymphocytes which fixed C3 fragments were T blasts that carried the CD3 marker and expressed IL-2R (CD25). CD4 and CD8 cells were represented with equal frequency in the C3-fragment fixing and C3-fragment negative populations. Pre-exposure of the MLC-activated lymphocytes to human serum increased their cytotoxic capacity toward CR type 2-carrying targets. The enhanced lysis was abrogated by F(ab)2 rabbit anti-human C3d or rabbit anti-CR type 2 antibodies. The results suggest that the C3 fragments fixed on the lymphocytes bind to CR on the targets and elevate the avidity of binding between the two interacting cells. This was also indicated by an increase in the frequency of lymphocyte-target conjugates.     

The role of insulin in the response of murine T lymphocytes to mitogenic stimulation in vitro

The ability of insulin to influence the responsiveness of murine T lymphocytes in a culture system containing a serum substitute was documented. The presence of insulin was found to enhance the concanavalin A (Con A) reactivity of the lymphocytes. Once the cells were activated by a short-term exposure to Con A, insulin was capable of replacing Con A for the continued stimulation of the cells. This was true both for lymphocyte proliferation and for the generation of nonspecific cytotoxic T lymphocytes. The presence or absence of insulin was not found to influence the phytohemagglutinin responsiveness of the T lymphocytes. Possible reasons for the observed results are discussed in relation to a proposed model for lymphocyte activation.     

Regulation of corneal collagenase production: stimulation of serially passaged stromal cells by blood mononuclear cells.

After three or more passages, stromal cell outgrowths from explants of normal rabbit corneas and from corneas previously burned with alkali did not produce collagenase until they were stimulated by the addition of rabbit blood-derived mononuclear cells or media conditioned by them. Normal stromal cells required stimulation by lymphocytes or their products from alkali-burned animals, whereas those from alkali-burned corneas were stimulated by lymphocytes from either normal or alkali-burned rabbits.     

Characterization of a low molecular weight suppressor of lymphocyte proliferation from guinea pig L2C leukemia cells

Conditioned medium (CM) from 24-hr culture of guinea pig L2C B lymphoblastic leukemia cells contained an inhibitor(s) of mitogen- and antigen-stimulated proliferation of syngeneic (strain 2 guinea pigs), allogeneic (Hartley guinea pigs), and xenogeneic (Balb/c mouse, NZW rabbit) lymphocytes. The proliferation of several lymphoid and nonlymphoid cell lines also was inhibited in the presence of CM. The inhibitor(s) in CM was not toxic to any of the cultures studied. CM inhibited the mitogen-stimulated proliferation of lymphocytes when added to cultures up to 52 hr after addition of mitogen. Normal responsiveness to mitogens could be restored by washing the CM-treated lymphocytes with medium during the first 6 hr of culture. The addition of exogenous IL-2 to lymphocyte cultures did not overcome the CM-mediated suppression of mitogen- or antigen-stimulated proliferation. CM also inhibited the IL-2-dependent proliferation of murine CTLL-2 cells. Preincubation of guinea pig lymphocytes in CM did not inhibit the capacity of these cells to release IL-2 after exposure to mitogen. The antiproliferative activity of CM was stable to heating at low pH (100 degrees C, 10 min, pH 4.0), was resistant to treatment with papain, pronase, DNase, and RNase and did not bind to Con A-Sepharose. Incubation of the L2C cells in indomethacin did not inhibit the release of the inhibitor(s). The inhibitor(s) in CM had an apparent molecular weight of 500-3500 Da as determined by dialysis and ultrafiltration analysis. The inhibitory activity was recovered in the organic phase after extraction with chloroform:methanol and eluted distinct from the thymidine standard after gel filtration on Sephadex-G 25. These data suggest that the inhibitor(s) in CM is a nonspecific, low molecular weight, lipid-like component (not prostaglandin) that exerts its antiproliferative effects subsequent to cell activation. The inhibitor(s) did not appear to suppress other biologic functions associated with activation, such as IL-2 secretion. The inhibitor in CM may be important in promoting tumor survival in vivo by suppressing potential anti-tumor cellular immune responsiveness.     

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.     

A microtechnique for PHA transformation of 5000 separated lymphoyctes

A microtechnique for studying phytohemagglutinin (PHA) responsiveness of 5000 separated human peripheral blood lymphocytes is described. Cells were distributed in conical-bottom microtiter wells for 3- and 5-day culture periods, after which stimulation was measured by incorporation of tritiated thymidine into DNA. Peak stimulation occurred over a narrow PHA dose range. More pronounced PHA stimulation was noted in 5-day cultures than in 3-day cultures using this technique, while the reverse was true for standard technique (500,000 lymphocytes). This microtechnique enables one to study PHA-induced proliferation of an extremely small number of separated human lymphocytes obtained not only from blood, but also from cellular compartments where lymphocytes are found in limited quantity.     

Fractionation of lymphocytes involved in the generation of cell-mediated cytotoxicity over insolubilized conjugated histamine columns.

Spleen cells from normal BALB/c mice were cultured in vitro with irradiated C57BL/6 stimulating cells. Five days later the T cell-mediated cytotoxic activity of the effector cells was assessed with a 51Cr-release assay that used H-2bEL-4 tumor cells as targets. Before the BALB/c responding lymphocytes were sensitized they were fractionated by passing the spleen cells over insolubilized histamine rabbit serum albumin Sepharose columns (H-RSA-S) or over rabbit serum albumin Sepharose (RSA-S) control columns. Fractionation of cells over the H-RSA-S columns depleted or significantly reduced the cytotoxic potential of the unretained cells. All cytotoxic potential was recovered when the cells that adhered to the H-RSA-S were eluted from the columns. In contrast, no effect on responsiveness was detected after the cells had been fractionated over the control column. The loss of response potential by the cells that did not adhere to H-RSA-S could not be accounted for by removal of macrophages nor by the concentration of cells with suppressor activity in the effluent. These cell fractionation studies raise the possiblity but do not prove that cytotoxic precursor cells may express amine receptors that could be responsible for their retention by insolubilized histamine columns.     

Stimulation of mitogenic responses in human peripheral blood lymphocytes by lipopolysaccharide: serum and T helper cell requirements.

Stimulation of human peripheral blood lymphocytes by lipopolysaccharide (LPS) was studied by the incorporation of 3H-thymidine. Peak stimulation occurred at 7 to 9 days over a broad range of LPS concentrations. Both Escherichia coli and Salmonella typhimurium LPS were effective mitogens with S. typhimurium having slightly higher activity. There was a strict serum requirement; pooled fresh frozen human serum was found to best support stimulation. In fetal calf serum, LPS caused a reduction in culture-induced stimulation. Cell separation procedures were employed in order to study the nature of the responding cell population. It was found that only non-T cells were stimulated by LPS, but in order for maximal stimulation to occur there was a requirement for helper T cells.     

Release of E-rosette augmenting factor (E-RAF) after stimulation of human leukocytes with mitogens or antigens.

Stimulation of human peripheral blood leukocytes (HPBL) with mitogens such as phytohemagglutinin or concanavalin A increases the total number of lymphocytes that form rosettes with sheep erythrocytes. A similar effect is seen when HPBL from skin test-positive, but not skin test-negative, donors are stimulated by a specific antigen. It was also found that the culture fluids from mitogen-stimulated lymphocytes contained a substance that significantly increased the percentage of E-rosette forming cells (85 to 95%) over that of control culture fluids (40%). A similar phenomenon was also observed with supernatant fluids derived from antigenic stimulation of cells from skin test-positive donors, but not from skin test-negative subjects. The factor that produces this effect has been designated E-rosette augmenting factor (E-RAF). It is nondialyzable; it appears in the supernatants of antigen or mitogen-stimulated cells within 12 hr; and its production is not blocked by mitomycin C. It is produced by cells that do not adhere to glass wool columns and by mitogen-stimulated thymocytes. Sephadex G-100 chromatography showed that mitogen-induced E-RAF eluted from the column in advance of antigen-induced E-RAF. E-RAF has many properties that are characteristic of lymphokines.     

In vitro studies of the rabbit immune system. VI. Rabbit anti-mouse cytotoxic T effector cells are inhibited by anti-rabbit T cell serum in the absence of complement.

Xenogeneic rabbit anti-mouse cell-mediated cytotoxic activity could be generated by culturing lymphoid cells from mesenteric lymph nodes (MLN), spleen, or peripheral blood of rabbits primed 2 to 8 weeks earlier with mouse tumor or spleen cells. MLN cells, which provided the best source of activity after being cultured with 5 to 10 X 10(6) mitomycin C-treated mouse spleen cells for 4 to 6 days, produced 30 to 90% specific isotope release after 4 to 7 hr incubation with 15Cr-labeled tumor target cells. Xenogeneic cytotoxic activity was primarily H-2 specific and could not be blocked by immune complexes but was abrogated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) before or after culture. Therefore, the activity appeared to be mediated by cytotoxic T lymphocytes (CTL). Furthermore, ATS without C abrogated cytotoxic activity when included in the CTL assay at concentrations of 5 to 15 microliter/10(7) effector cells. The inhibitory activity of ATS was directed to the rabbit effector population and could be absorbed completely by rabbit thymocytes. Antisera to mouse T cells with comparable cytolytic activity in the presence of C did not inhibit murine allogeneic CTL.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

B cells with or without C3 receptor: Murine B-cell subsets highly and lowly responsive to anti-Ig stimulation

Bone marrow-derived lymphocytes (B cells) with or without receptors for a third component of complement (CR) were studied in their responsiveness to the F(ab′)₂ fragment of antiimmunoglobulins (anti-Ig). Spleen cells from C57BL/6J mice were fractionated by the centrifugation over Ficoll-Hypaque density gradient after they were rosetted with erythrocyte-antibody complement complexes. The cells in the interface fraction responded poorly to anti-Ig, while the cells in the pellet fraction responded well. The low responsiveness of CR(?) B cells was confirmed by assaying the responsiveness of cells passed through a Sephadex G-10-complement column. Reduced response of CR(?) B cells could not be explained by the depletion of helper or accessory cells. The relationship between CR, B-cell differentiation and proliferative capacity of B cells is discussed.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. I. Activation by tuberculin and by Staphylococcus filtrate

Normal lymphocytes activated by mitogens such as phytohemagglutinin (PHA), by Staphylococcus filtrate (SF), or lymphocytes from sensitized individuals stimulated by antigen (PPD, etc.) are cytotoxic to tissue culture cells of different origins. In this and the following paper, the results of a detailed quantitative analysis of the specificity of this cytotoxic reaction are presented. Effector cells were human or mouse lymphocytes, activated by PHA, SF, PPD, or serum factors in the culture medium. Cells from established cell lines of human, mouse, hamster, or rabbit origin, or primary human or rat embryonic fibroblasts were used as target cells. Lysis was quantitated by release of ⁵¹Cr from labeled target cells.Purified human blood lymphocytes, activated by PPD, SF, or otherwise, preferentially damaged allogeneic target cells. Lysis of xenogeneic target cells was weak or did not occur. A close correlation was noted between target cell destruction and blastoid transformation of the lymphocytes, but the slope of the regression lines of xenogeneic cytotoxicity was much smaller than that of allogeneic cytotoxicity when plotted as a function of blastoid transformation.Lymph node or spleen cells from CBA mice were stimulated by PPD to transformation and DNA synthesis. CBA lymphocytes also showed an increased degree of blast transformation in medium containing fetal calf serum or certain batches of fresh human serum. Mouse lymphocytes activated in these ways damaged allogeneic L cells but had no effects on xenogeneic Chang cells.These results indicate that lymphocytes activated by various means preferentially damage target cells from their own species. The recognition mechanisms which determine the specificity of the reactions are not known.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

Antigen-antibody complex binding and cell interaction in stimulating normal rabbit lymphocytes

Complexes of antigen with specific antibody have been shown to enhance or suppress the specific antibody response in vivo. In vitro, antigen-antibody (Ag-Ab) complexes prepared in a slight antigen excess with rabbit antibody induced proliferation of unprimed rabbit lymphocytes. The Ag-Ab stimulated cells from a number of different normal lymphoid organs, including peripheral blood, bone marrow, spleen, and lymph node, but not thymus. Cells exposed to Ag-Ab for 1 hr and washed, bound Ag-Ab through Fc and complement receptors (CR), but were not induced to proliferate unless additional Ag-Ab was added. Specific antigen, which was otherwise unstimulatory, interacted with Ag-Ab-coated cells to activate them, probably through the cross-linking of membrane-bound ligands. Proliferation stimulated by Ag-Ab involved the interaction of three bone marrow cell subpopulations; a macrophage-enriched, a B-cell-enriched, and an mIgM- cell-enriched population. The separated subpopulations were poorly responsive to Ag-Ab stimulation, even though Ag-Ab bound to cells in each of the populations. Low levels of responsiveness to Ag-Ab also resulted when any two of the three subpopulations were combined. Only when all three subpopulations were mixed, was stimulation equivalent to the levels of stimulation reached by unseparated cells.