Showing papers in "Journal of Immunology in 1978"

T Cell Growth Factor: Parameters of Production and a Quantitative Microassay for Activity

Abstract: Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated thymidine incorporation of continuous murine tumorspecific cytotoxic T cell lines (CTLL). The microassay requires microliter quantities of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.

Autoantibody to a Nuclear Antigen in Proliferating Cells

Abstract: This study reports the identification of an autoantibody in the sera of some patients with systemic lupus erythematosus that reacted with nuclear antigen(s) of proliferating cells. The autoantibody was initially detected by the observation that it did not react in immunofluorescence with nuclei of renal tubular or glomerular cells, nor with hepatic parenchymal cells, but only reacted with scattered cells in the interstitial tissue of these two organs. In contrast, many lymphocytes in lymph node follicles, spleen, and thymus reacted with this antinuclear antibody. Normal peripheral blood lymphocytes did not show nuclear staining but after mitogenic stimulation, 20% of cells became positive. Nuclear staining was not restricted to lymphocytes but was also observed in several tissue culture cells lines such as Hep-2 cells (human epithelial carcinoma), Ehrlich ascites tumor cells, and baby hamster kidney cells. The reactive nuclear antigen(s) was soluble in physiologic saline and reacted with serum autoantibody to give a precipitin line in immunodiffusion that was immunologically distinct from DNA and other known nuclear antigen-antibody precipitating systems. Autoantibodies to proliferating cell nuclear antigen(s) might serve as useful biologic markers to study stimulated lymphocytes and other proliferating cells.

Immunologic functions of Ia-bearing epidermal Langerhans cells

Abstract: Langerhans cells (LC) constitute a morphologically well-characterized minor subpopulation of the mammalian epidermis whose functional role is still a matter of conjecture. The hypothesis that LC represent an epidermal equivalent to cells of the monocyte-macrophage-histiocyte series is supported by the recent observations that in humans and guinea pigs LC are the only epidermal cells that express Fc-IgG receptors, C3 receptors, and Ia antigens. Using inbred strain 2 and strain 13 guinea pigs, we investigated in this study whether LC can mediate the same immunologic functions as Ia-bearing macrophages. LC-enriched and LC-depleted epidermal cells were prepared by separation of Fc-IgG rosetting epidermal cells on density gradients. When both populations were tested for the biosynthesis of alloantigens by immunoprecipitation techniques, Ia antigen synthesis was restricted to the LC-enriched fraction. Functional studies demonstrated that antigen-pulsed LC-enriched epidermal cells induce a proliferative response in immune T cells that is comparable in magnitude to that seen with macrophages. Moreover, effective presentation of immunologically relevant antigen requires syngeneity between LC-enriched epidermal cells and responder lymphocytes. In the mixed leukocyte reaction (MLR), LC-enriched epidermal cells were as effective stimulators as macrophages. LC-depleted epidermal cells, by contrast, induced little or no stimulation in both assay systems. Both the antigen-presenting and the MLR-stimulatory capacities of LC-enriched epidermal cells could be abrogated by pretreatment with anti-Ia sera and complement. The presence in the epidermis of Ia-bearing LC, capable of mediating the immunologic functions of Ia-bearing macrophages, has important clinical implications with regard to the role of LC as sensitizing cells in both contact hypersensitivity and skin graft rejection.

The Activation of Mononuclear Phagocytes: Fact, Fancy, and Future

Abstract: Activation revisited. Since the time of Metchinkoff (1) and his contemporaries it has been clear that tissue macrophages exist in various states of activity. One reads about the resting-wandering cells of the connective tissue, arising from the mesenchyme and stimulated by the products of local injury. Such observations performed with the light microscope, vital dyes, and keen insight have in the past two decades been expanded with our increasing knowledge of the mononuclear phagocyte system (2). In the recent past the term “activated macrophage” has largely been relegated to the bactericidal process and to the role of these cells in cell-mediated immunity. Unraveled largely through the imaginative in vivo studies of Mackaness and North and their colleagues (3, 4), the two cell nature of the process, the effector role of the macrophage, and the unique sensitizing qualities of viable organisms were elucidated. Yet, it is apparent when one scans the varied roles of tissue macrophages in the physiology and pathology of mammals that this is a much restricted definition.

Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions.

Abstract: Human C5a was maximally active over a narrow concentration range of 2 to 6 × 10-7 M as estimated in rabbits by utilizing a skin window assay technique (Otani and Hugli, 1977). Human C5ades Arg also stimulated neutrophil accumulation under simulated in vivo conditions but at a slightly higher concentration range of 0.2 to 1.2 × 10-6 M. The C5ades Arg did not require added serum to produce an apparent leukotactic response in vivo, but, as in vitro, activity was not observed at levels above or below the effective concentration ranges indicated for C5a and C5ades Arg C3a was inactive over a concentration range of 5.5 × 10-9 to 5.5 × 10-6 M as an attractant for rabbit neutrophils in the skin window assay. The concentrations of C5a or C5ades Arg that are required to induce chemotaxis under simulated in vivo conditions correspond to the potential serum levels of these factors (e.g., 4 to 5 × 10-7 M). It is suggested from our quantitative measurements that the predominant chemotactic activity generated in human serum during C activation is associated with the C5ades Arg molecule. Therefore, C5ades Arg exhibits the potential for playing a predominant functional role under physiologic conditions as a major humoral chemotactic factor.

A hypothesis to relate the specificity of T lymphocytes and the activity of I region-specific Ir genes in macrophages and B lymphocytes.

Abstract: Ever since the discovery of specific immune response (Ir) genes in the I regions of the major histocompatibility complex (MHC) of mammals (1), certain characteristic features of the phenomena they control presented a considerable challenge: 1)H-linked Ir gene control has been observed only for thymus-dependent antigens that are proteins or polypeptides, and is concerned with T cell responses such as the stimulation of delayed-type hypersensitivity (DTH) and specific helper T cells (1, 2). 2)The specificity of Ir gene function is remarkable and distinguishes readily between antigens with different amino acid primary sequences (3–6). This has been demonstrated for synthetic antigens and also for native antigens. It is nevertheless remarkably distinct from the specificity of the T cell responses themselves (7), a matter of considerable concern to me when we initially proposed that the Ir gene product could be the elusive T cell receptor (1).

Subclass restriction of murine anti-carbohydrate antibodies.

Abstract: Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin

Biochemical Criteria for Activated Macrophages

Abstract: The designation “activated macrophage” was originally introduced to describe the state of macrophages that have an enhanced ability to phagocytize microorganisms and exert antimicrobial activity. This effect was found to be dependent upon particular infections and involves the participation of lymphocytes (1, 2). “Activation” has a specific immunologic basis, but its expression is nonspecific. Mackaness has written lucidly about activated macrophages and has referred readers back to the concepts of Lurie in tuberculosis, and further to the fons et origo of thinking in this context, i.e., to concepts of Metchnikoff (3). The latter9s oft quoted phrase regarding a “perfecting of the phagocytic and digestive powers of the leukocytes” might well be, broadly speaking, synonymous with “activation.” Once it was determined that macrophages taken from animals at some appropriate time after infection, for example, with Listerria or Bacillus Calmette-Guerin 2 (BCG), were superior to control cells with respect to their antimicrobial powers, it was natural for inquiry to be made into the differences between such cells and normal (control) cells, in terms of their morphology, cellular chemistry, and biochemical attributes.

The concept of the activated macrophage.

Abstract: The term “activated macrophage” was introduced into the literature and employed extensively by Mackaness in the 1960s to describe the enhanced microbicidal activity of macrophages from animals with acquired immunity to infection with facultative, intracellular bacterial parasites. The notion that macrophages need to undergo functional modification in order to express resistance to infection with certain bacteria can be traced back to the writings of Metchnikoff at the turn of the century (1). It was subsequent studies of tuberculosis more than of any other infectious disease, however, that provided the evidence for the concept of a mechanism of cellular immunity based on the capacity of macrophages to acquire and to intrinsically express increased microbicidal mechanisms. The knowledge a) that a major visible aspect of the host response to tuberculosis consisted of the accumulation of macrophages at infective foci to form tubercles, b) that the tubercle bacillus is invariably engaged by and harbored inside macrophages, c) that secondary infection results in an accelerated rate of tubercle formation and in the destruction of the tubercle bacilli therein, and d) that all of these events proceed apparently without the participation of humoral antibody were sound reasons for postulating a mechanism of cellular immunity based on the intrinsic microbicidal powers of the macrophage.

Characterization of lymphocyte-activating factor (LAF) produced by the macrophage cell line, P388D1. I. Enhancement of LAF production by activated T lymphocytes.

Abstract: Based on the results of these studies, it appears that P388D1 cells are an excellent source of LAF for both biologic and biochemical studies

Lymphocyte-Mediated Activation of Fibroblast Proliferation and Collagen Production

Abstract: Among the many activities of antigen-triggered lymphocytes may be the control of fibroblast function. Thymus-dependent lymphocytes challenged with the specific antigen, dinitrophenylated ovalbumin or tetanus toxoid produced a nondialyzable factor(s) capable of causing dermal fibroblasts to undergo DNA synthesis. These fibroblasts, which exhibit basal proliferative levels in the absence of serum, responded to the lymphocyte factor with maximal thymidine incorporation at 48 to 72 hr. In addition, these activated fibroblasts significantly increased their production of protein of both the collagenous and noncollagenous types. This increase in protein synthesis preceded maximal proliferation. Thus, the fibroplasia consisting of increased numbers of fibroblasts and of increased collagen deposition associated with chronic inflammatory diseases may be the direct consequence of a specific antigenic challenge.

Murine B Cell Leukemia Line with Inducible Surface Immunoglobulin Expression

Abstract: A chemically induced leukemia of BDF 1 mice, designated 70Z/3, was adapted to tissue culture in our laboratory A minority of these cells displayed surface immunoglobulin (sIg) as detected by immunofluorescence with rhodamine-labeled class (IgM)-specific antibodies Addition of the B cell mitogen, LPS, to the cultures induced sIg expression on all of these cells The kinetics of this transition were dependent on the dose of LPS As little as 01 µg/ml induced sIg on >97% of the cells within 36 hr DxS also induced sIg whereas other mitogens (Con A, PPD) or inducing agents (DMSO, dimethyl formamide, butyric acid), tested over a wide range of concentrations, failed to induce sIg expression The cells bear H-2 antigens but lack IgD, IgG, IgA, Ia, and Thy 12 Exposure to LPS had no effect on the presence or absence of these structures A small percentage of cells possessed receptors for complement and for the Fc portion of immunoglobulin Cytoplasmic IgM was detectable within all of the cells, and constitutive production of small quantities of IgM was confirmed by SDS-polyacrylamide gel electrophoresis of serologic precipitates of cell lysates after pulsing with radioactive amino acids Using similar methods, we failed to detect active secretion of immunoglobulin Thus, this cell line has properties similar to cytoplasmic Ig + , surface Ig - cells found in immature tissues and in bone marrow of mice and humans that are thought to be immediate precursors of sIg + B lymphocytes It may provide a model for studying the mechanism of LPS activation and the molecular events associated with externalization of cell surface receptors

Immunoregulatory Aberrations in Systemic Lupus Erythematosus

Abstract: This study was undertaken to delineate the nature of the immunoregulatory defect in patients with systemic lupus erythematosus (SLE). Thirty-eight SLE patients were studied and it was found that in addition to an absolute T lymphocytopenia, these patients had a selective proportional and quantitative deficiency (P G ). This T cell subpopulation has been previously demonstrated to be the suppressor cell population in pokeweed mitogen (PWM)-driven intracytoplasmic Ig production. T cells with a receptor for IgM (T M ) which have been shown to be the helper T cells for Ig production were not significantly different from normals in proportion or absolute numbers. By employing a PWM-induced plaque-forming cell (PFC) assay against sheep red blood cells (SRBC), it was shown that SLE patients had a markedly reduced response of 18 (±4.2) PFC/10 6 lymphocytes compared to normals with 153 (±18.4) PFC/10 6 lymphocytes (P in vivo polyclonal activation associated with the autoimmune state. SLE patients had a marked defect in the ability of their lymphocytes after Con A activation to generate suppressor cells of the PWM-induced PFC response, whereas their ability to be suppressed by suppressor stimuli appeared intact. Despite considerable variability from patient to patient, lymphocytes from SLE patients in general had adequate T helper cell function and could be helped by normal allogeneic T helper cells. Thus, this study demonstrates that SLE patients have a quantitative defect in a subpopulation of T cells with suppressor capability. In addition, they have a defect in the ability to generate suppressor cell function upon appropriate activation. These findings may lend further insight into the understanding of the immunoregulatory defect in SLE.

Characterization of the Effects of Endotoxin on Macrophage Tumor Cell Killing

Abstract: These studies demonstrate the potent effect of LPS on the induction of macrophage-mediated tumor cell killing and show, by using pure populations of cloned macrophages as effector cells, that LPS can act in the complete absence of contaminating lymphocytes. Macrophage differentiation toward the tumoricidal state parallels the responsiveness of macrophages to LPS. Normal macrophages will not kill even in the presence of 10 µg/ml LPS; peptone-induced normal macrophages (stimulated macrophages) are made tumoricidal by ≥500 mg/ml LPS; and nontumoricidal BCG-activated macrophages are made to kill tumor cells by 0.5 to 1.0 ng/ml LPS. The LPS effect is reproduced by lipid A. The effect is abolished by alkaline treatment of LPS or by the presence of polymyxin B. Nontumoricidal BCG-activated macrophages from the lipid A-nonresponder mouse strain C3H/HeJ require 250 times more LPS to become tumoricidal than do nontumoricidal BCG-activated macrophages from the C3H/HeN strain. LPS acts synergistically with MAF-rich peritoneal cell supernatants in causing macrophages to kill tumor cells. Although LPS can elicit MAF production by lymphocytes, it can also act directly on cells of lymphocyte-free macrophage colonies to render them tumoricidal. Reagents are frequently contaminated with LPS, and unless this is noted, valid interpretation of experiments investigating macrophage tumor cell killing is difficult. Means of detecting, detoxifying, and eliminating the LPS of experimental agents are discussed.

Increased Spontaneous Polyclonal Activation of B Lymphocytes in Mice with Spontaneous Autoimmune Disease

Abstract: Early in life, mice of four kinds [NZB, (NZB X NZW)F1, MRL/1, and male BXSB] with autoimmune disease spontaneously produced far more (greater than 3 S.D.) anti-hapten antibody-forming cells in spleens and greater concentrations of anti-hapten antibodies in sera than immunologically normal strains of mice (AKR, BALB/c, C57BL/6, DBA/1-J, DBA/2J, LG/J, 129, NZW, and female BXSB). This increased nonspecific antibody production by the abnormal animals' B cells correlated well with the spontaneous development of anti-single-stranded DNA antibodies, but not with serum levels of the viral envelope glycoprotein, gp70. These results suggest that the spontaneous formation of autoantibodies in mice whose immunologic disorder is manifested by a lupus-like disease may result from polyclonal activation of B cells by endogenous or exogenous B cell activators.

Intracellular Control of Human Neutrophil Secretion I. C5a-Induced Stimulus-Specific Desensitization and the Effects of Cytochalasin B

Abstract: Human neutrophils released the granule constituents myeloperoxidase and lysozyme, but not the cytoplasmic enzyme lactic dehydrogenase, when pretreated with cytochalasin B and stimulated with purified human C5a. Prior exposure to C5a before the cytochalasin B, however, abrogated the subsequent secretory process. Interaction of neutrophils with C5a was shown to result in a concentration-dependent rapid desensitization that could not be overcome by later addition of cytochalasin B or of cytochalasin B and C5a. The effect was relatively stimulus specific in that neutrophils desensitized in this manner could be induced to release granule enzymes by casein or by complement-coated zymosan particles. Cytochalasin B effects on neutrophils appear to mimic those of surface binding of soluble stimuli such as C5a and immune complexes. It is suggested that desensitization in concert with surface stimulation may represent an important intracellular mechanism for limiting neutrophil secretion.

The Genetic Mapping of a Defective Lps Response Gene in C3H/HeJ Mice

Abstract: The expression of a defective LPS response gene Lps and the major urinary protein (Mup-1) are concordantly inherited in backcross (C3H/HeJ x C57BL/6J)F1 x C3H/HeJ mice, indicating genetic linkage of these loci. Mup-1 is known to be linked to the brown coat color locus on chromosome 4 in mice; thus Lps can now be assigned to chromosome 4. A value of 0.06 +/- 0.02 has been estimated for the recombination frequency between Mup-1 and Lps. We have used the polysyndactyly (Ps) mutation further to localize Lps on chromosome 4. Lps is located between the Mup-1 and Ps loci.

Potentiation of mast cell mediator release by adenosine.

Abstract: Since extracellular adenosine can alter adenylate cyclase activity and cell function in a variety of tissues, the effect of adenosine on mediator release was studied with isolated rat mast cells. Adenosine did not alter the rate of spontaneous release, but histamine release induced by anti-IgE, concanavalin A (Con A), 48/80, or the calcium ionophore A23187 was markedly enhanced by 1 nM to 0.1 mM adenosine. Mast cells preincubated in the presence of 10 µM adenosine and then challenged with 0.1 µg/ml of A23187 released 71.5% (± 7.4 SEM) of the cellular histamine compared to a 38.2% (± 3.0 SEM) release from control cells. Adenosine caused a 3.0-fold (± 0.2 SEM) increase in the rate of release occurring 30 to 60 sec after A23187 addition, but did not change the duration of mediator release. Adenosine did not appear to decrease the minimum concentration of ionophore required to stimulate release. The poorly metabolized adenosine analog 2-chloroadenosine (100 µM) potentiated release approximately 2-fold whereas dipyridamole, an inhibitor of mast cell uptake of adenosine, did not inhibit adenosine potentiation suggesting that adenosine actions on mast cells may be mediated by direct activation of cell-surface receptors. Adenosine modestly potentiated the fall in cyclic AMP levels occurring 30 to 60 sec after ionophore challenge, raising the possibility that potentiation may be mediated by alterations in cyclic AMP metabolism. Mast cells suspended in media containing 1 mM EGTA and no added calcium demonstrated adenosine potentiation of A23187-induced release indicating that the adenosine effect was not mediated by increased translocation of calcium into the cells. Adenosine potentiation was completely blocked by 100 µM theophylline, a known inhibitor of adenosine binding, with 50% inhibition of release occurring at 3 µM theophylline. These studies indicate that adenosine may be an important modulator of mast cell function in complex tissues and that part of the pharmacologic action of theophylline in human bronchial asthma may be through inhibition of adenosine binding, rather than direct effects on cyclic AMP metabolism.

Target-effector interaction in the natural killer (NK) cell system. II. The isolation of NK cells and studies on the mechanism of killing

Abstract: We have previously shown that a subpopulation of nylon wool nonadherent, lymphoid cells in normal mice binds selectively to tumor cell targets that are sensitive to killing by natural killer (NK) cells. In the present report these target-binding cells (TBC) were enriched or depleted by velocity sedimentation and the isolation of single target-effector conjugates indicated that the majority of TBC killed their targets. Furthermore, the frequency of TBC correlated well (r = 0.86) with lysis in a population during kinetics experiments. TBC resembled small, resting lymphocytes with membrane specializations in the area of target cell contact as indicated by electron microscopy and cytochemical techniques. Target cell binding occurred before lysis in kinetics studies and regenerated in parallel with the lytic potential of a trypsinized population devoid of surface Ig or θ-bearing cells. Cell contact was prevented in the presence of EDTA whereas metabolic inhibitors or inhibitors of serine proteases suppressed lysis with no effect on the frequency of TBC. Conversely, an interferon-inducing agent elevated NK-mediated cytolysis with no effect on the level of TBC. These results suggest that i) the majority of TBC that we detect represents the NK cell and ii) lysis and binding are independently controlled events. A tentative model of NK-mediated lysis is proposed.

Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer's patches after oral administration of sheep erythrocytes and their systemic migration.

Abstract: Rats given 10(10) sheep erythrocytes (SRBC) orally were found to contain specific suppressor cells to SRBC in their Peyer's patches (PP) and mesenteric lymph nodes (MLN) after 2 days of feeding. After 4 days of feeding, similar suppressor cells were found in the thymus and spleen, but they were missing in the PP or MLN. These suppressor cells effectively blocked IgM and IgG plaque-forming cell responses to SRBC in Mishell-Dutton cultures and delayed-type-hypersensitivity responses to SRBC when transferred to syngeneic recipients, but they did not affect responses to horse erythrocytes. The orally induced specific suppressor cells appeared to be T2 cells since their activity was eliminated by in vivo treatment of SRBC-fed rats with anti-rat lymphocyte serum but not by adult thymectomy. Because carrageenan partially relieved the suppression observed in culture, the actual suppressive mechanism may also involve a macrophage.

Macrophage Activation for Tumor Cytotoxicity: Development of Macrophage Cytotoxic Activity Requires Completion of A Sequence of Short-Lived Intermediary Reactions

Abstract: Macrophages from endotoxin (LPS)-unresponsive C3H/HeJ mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that induce cytotoxicity with cells from LPS-responsive C3H/HeN mice. Under certain conditions, however, these macrophages could become tumoricidal: macrophages from in vivo immune reactions such as those induced by BCG infection, but not cells from irritant-induced peritoneal exudates, developed full cytotoxic capability after further exposure in vitro to microgram per milliliter concentrations of LPS or to supernatants from antigen-stimulated leukocyte cultures (lymphokines). Capacity of macrophages from BCG-infected C3H/HeJ mice to become tumoricidal after LPS exposure was gradually lost with time in culture and was absent by 24 hr. Requirements for at least two activation stimuli for cytotoxic activity with C3H/HeJ macrophages could also be fulfilled entirely in vitro : cells cultured in lymphokines plus LPS were fully tumoricidal; macrophages cultured in either agent alone were not cytotoxic. Similar interactions between lymphokines and LPS could be demonstrated with macrophages from LPS-responsive C3H/HeN mice. Tumoricidal activity by C3H/HeN macrophages cultured in lymphokines plus nanogram per milliliter concentrations of LPS was considerably greater than that by cells cultured in either agent alone. LPS and lymphokines were synergistic in their action on macrophage cytotoxicity. The synergistic effect of LPS on cytotoxicity by lymphokine-activated macrophages was evident after a 15-min pulse and was not dependent upon fluid-phase LPS. Synergistic action, however, was dependent upon 1.) treatment sequence: LPS was active only if given simultaneously with or after lymphokine treatment; LPS exposure before lymphokine treatment was ineffective, and 2.) treatment interval: capacity of lymphokine-treated macrophages to express cytotoxic activity after LPS exposure decayed with time in culture and was lost by 24 hr. Thus, macrophage activation for tumor cytotoxicity in both C3H/HeJ and C3H/HeN mice was the final result of a cascade of short-lived intermediary reactions in a defined sequence. Tumoricidal activity of fully activated cells and responsiveness of each noncytotoxic precursor cell to activation stimuli were all short lived macrophage reactions. Present evidence suggests that none of these reactions was reversible. Completion of each state of macrophage activation was completely dependent upon the simultaneous presence of localized activation signals and macrophage precursors able to respond.

Serologic Typing of Rickettsiae of the Spotted Fever Group by Microimmunofluorescence

Abstract: The micro-immunofluorescence (micro-IF) method was used to type rickettsiae belonging to the spotted fever or typhus groups according to their surface antigens. Seventy-two strains of rickettsiae from diverse sources, with varying histories of laboratory manipulation and immunologic characterization by other methods, were cross-tested against their mouse antisera. Fifteen serologic patterns (serotypes) were observed, 12 associated with spotted fever-group (SFG) rickettsiae and three with typhus group (TG) organisms. The reaction patterns of strains within each serotype were homogeneous. Nine of the SFG serotypes and the three TG serotypes were characteristic for rickettsiae that have been classified by other biologic procedures as to species (classified serotypes). The classified SFG serotypes included Rickettsia rickettsii (R-like), R. rickettsii (Hlp-like), R. sibirica, R. parkeri, R. conorii, R. rhipicephali, R. montana, R. australis , and R. akari . The three TG serotypes included R. prowazekii, R. typhi , and R. canada . Three SFG serotypes included rickettsiae that have not been identified as to species. Each consisted of two or more strains isolated in different years from widely-spaced localities in the United States and were considered to be distinct serotypes, which for the time being are unclassified. They included two strains of rickettsiae isolated from Dermacentor occidentalis ticks in California, three strains recovered from D. parumapertus ticks in southwestern United States, and three strains obtained from Ixodes pacificus ticks in Oregon. Each of three other strains had patterns of reaction that differed from those of all other rickettsiae. However, they were single representatives and evidence derived from this study is not sufficient to consider these as comprising additional serotypes. The results obtained by micro-IF are in general agreement with other procedures for antigenic differentiation of rickettsiae belonging to the SFG and TG. Therefore, an immunologic basis for serologic classification of these rickettsiae is likely. Micro-IF should prove to be particularly useful for determining taxonomic and epidemiologic relationships among SFG rickettsiae because of its simplicity and general applicability.

Macrophage Ia Antigens I. Macrophage Populations Differ in Their Expression of Ia Antigens

Abstract: By indirect immunofluorescence and microcytotoxicity it was demonstrated that different populations of murine macrophages bear different amounts of Ia antigens on their membranes. At least three subpopulations could be distinguished: those that lack Ia antigens and predominate in peritoneal exudate; cells bearing I-A antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-C antigens that are a minor population in both spleen and peritoneum. Internal radioisotope labeling studies confirmed that the I region molecules are synthesized by the macrophages. It is suggested that these different macrophage subpopulations may play distinct roles in the immune response.

B Lymphocyte Precursors in Human Bone Marrow: An Analysis of Normal Individuals and Patients with Antibody-Deficiency States

Abstract: Lymphoid cells containing cytoplasmic IgM but lacking stable surface IgM are believed to be the direct precursors of B lymphocytes. We have characterized these pre-B cells in the bone marrow of normal individuals and patients with a variety of immunoglobulin deficiencies or hematologic disorders by using immunofluorescence and autoradiography. Pre-B cells comprised 5.8 +/- 5.7% of lymphoid cells in normal bone marrow. Eleven patients with infantile X-linked agammaglobulinemia (X-LA) lacked B lymphocytes but had a normal frequency (3.8 +/- 3.6%) of bone marrow pre-B cells. A smaller proportion of marrow pre-B cells from patients with X-LA were engaged in spontaneous DNA synthesis than was found for normal controls. In individuals other than the group with X-LA, the number of circulating B cells was positively correlated with the frequency of marrow pre-B cells. These results indicate that patients with X-LA have a defect in maturation of pre-B cells, and suggest that some patients with acquired B lymphocyte deficiency may have lost the capacity to generate pre-B cells from stem cells.

Enterically Induced Immunologic Tolerance I. Induction of Suppressor T Lymphocytes by Intragastric Administration of Soluble Proteins

Abstract: Specific immune unresponsiveness was induced in inbred mice (BDF1) by the administration of soluble ovalbumin (OVA) by gastric intubation. Anti-hapten (DNP) responses likewise were specifically diminished when animals were fed autologous carrier (OVA or keyhole limpet hemocyanin). Adoptive transfer of spleen cells demonstrated that the tolerant state could be maintained in irradiated recipient mice, and specific anergy could be transferred to normal recipient animals. Adoptive suppression was mediated by T lymphocytes, as demonstrated by nylon wool fractionation and susceptibility of the cells to anti-Thy 1.2 and complement. Transferred B cells had neither suppressive nor augmentative effects. Enteric administration of OVA also specifically diminished antigen-induced DNA synthesis of primed lymph node T cells, although suppressor cells were not identified in the lymph nodes per se.

Cell-mediated cytotoxicity against virus-infected target cells in humans. II. Interferon induction and activation of natural killer cells.

Abstract: The mechanisms by which human lymphocytes lyse virus-infected allogeneic fibroblast cultures were analyzed with particular consideration of the role of antiviral antibodies and interferon. Human cells infected with viruses were able to induce high levels of interferon upon contact with human lymphocytes. Interferon, whether produced by lymphocytes after direct infection with virus or induced upon exposure of lymphocytes to virus-infected fibroblasts, appeared to be responsible for enhancing the cytotoxic efficiency of the natural killer cell against the infected target. Activation of cytotoxic lymphocytes occurred as early as 6 hr after addition of interferon and increased up to 24 hr. Antibody-dependent cell-mediated cytotoxicity (Ab-CMC) could be easily induced by sensitization of infected target cells with antiviral antibodies and could be detected at 4 hr from the beginning of the cytotoxic test, before the effect of interferon on the natural killer cell was evident. However, the antibody-dependent effector cell was inactive after 4 hr of incubation. F(ab′)2 fragments of rabbit antihuman IgG completely inhibited Ab-CMC but did not at all affect the spontaneous cytotoxic activity of the effector cells against virus-infected target.

Beta-estradiol reduces natural killer cells in mice.

Abstract: beta-estradiol was administered to mice continuously by diffusion from a silastic tube that was implanted subcutaneously at 4 weeks of age. Four to 6 weeks of estrogen administration caused a substantial reduction in natural killer cell activity in the spleens from mice of either sex. Androgen (5alpha-dihydrotestosterone) did not. Castration of male or female mice did not affect natural killing and did not alter the effect of beta-estradiol. Estradiol did not affect natural killing in vitro and the loss of natural killing was not due to a soluble or a cellular suppressor of natural killing. The effects of estradiol were not dependent on the thymus, since estradiol reduced natural killing in mice that had been neonatally thymectomized. After removal of the estrogen implant, natural killing recovered over a period of 8 weeks. The loss of natural killing may reflect a loss of bone marrow secondary to estrogen-induced osteosclerosis.

Suppressor T Cells for IgE and IgG in Peyer's Patches of Mice Made Tolerant by the Oral Administration of Ovalbumin

Abstract: A single oral dose of ovalbumin (Ov) resulted in inhibition of IgE formation in mice subsequently immunized i.p. with Al(OH) 3 -Ov. Repeated feeding of Ov (on alternate days for 2 weeks) induced the formation of detectable suppressor cells in Peyer9s patches and spleen. Suppression was demonstrated by the ability of adoptively transferred Peyer9s patch or splenic lymphocytes from Ov-fed tolerant mice to inhibit IgE formation in Ov-immunized syngeneic recipients. Suppressor cells could be induced by feeding mice as little as 100 µg of Ov on alternate days for 2 weeks. Suppression was specific and Peyer9s patch lymphocytes were shown to be more effective suppressors than splenic lymphocytes.

Suppressor Adherent Cells in Human Tuberculosis

Abstract: Patients with newly diagnosed active pulmonary tuberculosis were divided into high and low responder groups on the basis of 3 H-thymidine incorporation of peripheral blood mononuclear cells (PB MNC) induced by tuberculin-purified protein derivative (PPD). The group of patients with low tuberculin responses was characterized by anergy to tuberculin skin testing and larger numbers of circulating monocytes than the high responders. The groups were comparable in the extent of pulmonary tuberculosis and other clinical parameters. PB MNC from tuberculin low responders had normal phytohemagglutinin (PHA) but depressed streptokinase-streptodornase (SKSD), Candida antigen, and tetanus toxoid-induced 3 H-thymidine incorporation. Depletion of adherent cells resulted in a mean 24.4-fold enhancement of T lymphocyte responses to PPD in the low responders as compared to a 2.9-fold increase in the high responders. The low response of PB MNC from low responders to nonmycobacterial antigens was not similarly augmented by adherent cell depletion. Adding back graded numbers of adherent cells to purified T lymphocyte populations resulted in greater depression of the PPD-induced proliferative response to the tuberculin low responders when compared to the high responders. These studies provide evidence for circulating suppressor adherent cells in tuberculosis patients with diminished responsiveness to PPD in vitro . This immunosuppression is not mediated by adherent cell supernatant products, nor does it involve T lymphocyte labile during culture. However, complex cellular interactions, possibly involving suppressor T cells, are implicated by the restriction of this phenomenon to PPD-induced lymphocyte activation and the depression of the PB MNC responses to nonmycobacterial antigens. The suppressor adherent cells defined in these studies may be relevant as a cause or consequence of infection in a subset of patients with tuberculosis.

Spontaneous Cell-Mediated Cytotoxicity in Humans: Role of Interferon and Immunoglobulins

Abstract: Human lymphocytes secrete high levels of interferon a few hours after being cultured with certain tumorderived or virus-transformed cell lines. Interferon and interferon inducers (e.g., viruses, inducer cell lines, synthetic inducers), after short incubation with lymphocytes, increase several-fold the cytotoxicity of human natural killer cells. When lymphocytes are tested as effector cells against interferon-inducing target cell lines in an 18-hr test of spontaneous cell-mediated cytotoxicity, the enhancing effect of the interferon released in the culture medium is responsible for 80 to 90% of the total cytotoxicity observed. The ability or inability of different target cell lines to induce interferon is responsible for the apparent difference in selectivity of the cytotoxicity against various targets when fresh lymphocytes, cultured lymphcoytes, or interferon-activated lymphocytes are used as effector cells. Moreover, some of the apparently specific results obtained in competitive assays with unlabeled target cells are also dependent on the ability or inability of the competitor and target cells to induce interferon. Interferon does not increase the antibody-dependent cytotoxicity mediated by human lymphocytes, which suggests that spontaneous and antibody-dependent cell-mediated cytotoxicity are mediated by different effector cells or that a unique class of effector cells can mediate cytotoxicity with two independent mechanisms. The inability of rabbit F(ab′) 2 fragment antihuman IgG to inhibit spontaneous cell-mediated cytotoxicity, renders unlikely the possibility that the activity of the human natural killer cells is dependent on cytophilic anti-target cell antibodies adsorbed in vivo to the effector lymphocytes.