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Publications and source records attributed to J S Abrams.
Compartment syndromes of the upper arm are reported rarely in the literature. The diagnosis requires a high index of suspicion. As with compartment syndromes elsewhere, prompt aggressive treatment is crucial in avoiding possible crippling sequelae.
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Production of IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, GM-CSF (granulocyte-macrophage colony stimulating factor) and macrophage colony stimulating factor by murine peripheral blood and spleen cells was analyzed following primary and secondary mitogen stimulation in vitro. Individual cytokine producing cells were detected by an intracytoplasmic staining technique. Cytokine production in cells from peripheral blood and spleen was comparable and more rapidly induced by calcium ionophore and phorbol 12-myristate 13-acetate than by concanavaling A. Restimulation in vitro induced both a swift production of cytokines and, for some cytokines, higher frequencies of producing cells. This was especially evident for IL-10 secreting cells, which increased 30-80 times in secondary responses. These analyses using the dual approaches of immunoenzymetric and fluorescent immunohistochemical techniques provide important evidence that cytokine induction kinetics can differ following primary or secondary stimulation.
Helminth infections in humans and animals are associated with strong T helper 2 (Th2) responses. To determine whether parasite-derived Ag preferentially expand a Th2-like cell population, a filter immunoplaque assay was used to enumerate the frequencies (F0) of PBMC and CD4(+)-enriched PBMC from individuals with helminth infections secreting selected cytokines in response to parasite-derived (PAg) and nonparasite antigens (NPAg). In 20 individuals with lymphatic filariasis, frequency analysis of PBMC secreting IL-4 and IFN-gamma indicated that the F0 of PAg-specific IL-4-secreting cells (geometric mean F0 (GM): 1/12,100) was 57-fold higher than the corresponding F0 of NPAg-reactive cells (GM: 1/692,000; p < 0.02). In marked contrast, the F0 of IFN-gamma-secreting cells responding to PAg (GM: 1/2,700) did not differ from those of cells specific for NAPg (GM: 1/3,400; p = 0.83). In another group of helminth-infected individuals, the F0 of highly enriched CD4+ cells secreting IL-4 and IL-5 in response to PAg (GMs: 1/2,600 and 1/5,600 CD4+ cells, respectively) were also found to be significantly higher than those specific for NPAg (GMs: 1/291,000 and 1/303,000 CD4+; p < 0.05 and p < 0.01, respectively), whereas the corresponding F0 of IFN-gamma- and granulocyte-macrophage-CSF-secreting cells were equivalent for PAg and NPag. Furthermore, the proportion of PAg-specific IL-4- and IL-5-secreting CD4+ cells relative to all cells secreting the given cytokine were approximately 29-fold higher than the proportion of NPAg-specific cells secreting these cytokines. Again, the corresponding proportions of Ag-specific IFN-gamma-and GM-CSF-secreting CD4+ cells were equivalent for PAg and NPAg. Thus, in this ex vivo system, a circulating population of IL-4- and IL-5-secreting (Th2-like) cells has been shown to exist in humans; PAg appears to expand these cells preferentially.
Recent data indicate a major role for IL-10 in suppressing immune and inflammatory reactions. To date, expression of human IL-10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL-6 expression. In this study we sought to determine whether non-hematopoietic human tumor cell lines produce IL-10 and, if so, what is the relationship between IL-10 and IL-6. Using ELISA, we determined IL-10 and IL-6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL-6 protein was secreted by 28 of the tumor cell lines; IL-10 was measurable in 15 cell lines. IL-6 secretion was maximal and most frequent in renal-cancer cell lines, while IL-10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL-10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio-active, as demonstrated in the mouse MC/9 mast-cell-line assay and in human mixed-lymphocyte reactions. In both assays, IL-10 bio-activity was neutralized by an anti-IL-10 monoclonal antibody. Expression of IL-6 and IL-10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL-1 alpha, and TNF-alpha strongly enhanced the release of IL-6 by RCC cells, but only marginally affected IL-10 production in colon-carcinoma cells. IL-10 secretion by colon-carcinoma cells was moderately stimulated by IFN-gamma and IL-4. Dexamethasone suppressed the release of IL-6, but had no inhibitory effect on IL-10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL-6 and/or IL-10, suggesting a role of these cytokines in solid-tumor development and anti-tumor immunity.
The results of this study, in which CD4 T cells were harvested from mice at various times during the course of a virulent Mycobacterium tuberculosis infection and examined for their secretion of cytokines during culture in vitro with bone marrow-derived macrophages presenting mycobacterial Ag, provide evidence for the existence of two separate waves of cytokine-producing CD4 cells. The first, which peaks at the time at which protective immunity was maximally expressed, was characterized as a IFN-gamma-secreting cell population that preferentially recognized macrophages presenting mycobacterial culture filtrate proteins, or that were infected with the live organism. A second population, which emerged 20 to 40 days later at a time when the infection had been contained, secreted IL-4 in response to the filtrate proteins, but also reacted particularly strongly to the 65-kDa (hsp60) heat shock protein molecule of M. tuberculosis. These data indicate that acquired immunity to tuberculosis infection involves the production of both Th1- and Th2-like cell populations that differ in terms of their kinetics of emergence and loss, and in terms of their Ag specificity.
IL-10 and IL-4 have been shown to exert an inhibitory effect on cell-mediated immune responses. Our previous studies of leprosy demonstrated that IL-10 and IL-4 mRNA were preferentially expressed in lesions from lepromatous patients, those immunologically unresponsive individuals that manifest widespread infection. To define more precisely the regulatory roles of these two cytokines in the immune response to infection, we studied in vitro responses to Mycobacterium leprae. M. leprae triggered IL-10 release from PBMC of patients and healthy donors; the predominant source of the IL-10 was found to be monocytes/macrophages. Stimulation of PBMC in the presence of neutralizing anti-IL-10 mAb indicated that endogenous IL-10 production inhibits PBMC proliferation and release of TNF-alpha, GM-CSF, and IFN-gamma. Paradoxically, studies using neutralizing anti-IL-4 mAb indicated that endogenous IL-4 production enhances PBMC proliferative responses most strikingly in lepromatous patients. We found that rIL-4 expanded CD8+ T cells from lepromatous patients in vitro. CD8+ T cells from lepromatous patients have been shown to suppress CD4+ T cell responses, in part by the release of IL-4. Our study indicated that endogenous IL-4 production inhibited IL-10 secretion and, concomitantly, increased TNF-alpha and GM-CSF release. The present data suggest that, on balance, IL-4 and IL-10 contribute to immunosuppression in human infectious disease.
Although Th2 helper cell clones produce IL-4 and IL-5, CD4+ T cells taken fresh from lymphoid organs of mice produce IL-2 and some IFN-gamma, but not IL-4 or IL-5. The exact parameters that enhance the synthesis of IL-4, IL-5, and particularly IL-10 from resting antigen-specific CD4+ T cells is not yet clear. We therefore examined the kinetics of, and the parameters that affect, the development of IL-4, IL-5, and IL-10 production in bulk populations of antigen-specific human CD4+ T cells. We demonstrated that in vitro stimulation of human peripheral blood lymphocytes with antigen (tetanus toxoid or a viral antigen, Varicella zoster) for several days resulted in the production of IL-2 and IFN-gamma, but little or no IL-4 or IL-5. This cytokine profile was observed even when CD4+ T cells from allergic donors were stimulated once in vitro with allergen (rye grass pollen, Lolium perenne I, or dust mite allergen, Dermatophagoides farinae). The observed cytokine profile reflected that of the vast majority of antigen-specific T cells, since we studied the response of bulk populations of CD4+ T cells rather than that of a "selected" number of antigen-specific T cell clones. Furthermore, we observed that human CD4+ T cells from either allergic or nonallergic individuals failed to produce significant quantities of IL-4, IL-5, or IL-10 even after several rounds of stimulation with soluble protein (nonallergen) antigens such as tetanus toxoid or Var. z. However, large quantities of these cytokines were produced when stimulation of the T cells occurred in the presence of rIL-4, but only after two stimulations in vitro with antigen over a period of greater than 7 days. In addition, substantial quantities of IL-4, IL-5, and IL-10 were produced by CD4+ T cells from allergic subjects in the absence of exogenous IL-4, but only after two stimulations in vitro with allergens such as rye grass pollen or dust mite allergen. These results indicate that the development of IL-4 and IL-5 synthesis occurs in peripheral blood CD4+ T cells in a stepwise fashion, first with the production of IL-2 and IFN-gamma, and later with the production of IL-4 and IL-5.(ABSTRACT TRUNCATED AT 250 WORDS)
Patients from across the spectrum of clinical manifestations of Leishmania chagasi infection were evaluated for in vitro correlates of immunity. Peripheral blood mononuclear cells were assayed for parasite-specific lymphoproliferation, cytokine generation, and the capacity to activate autologous macrophages to kill intracellular amastigotes. Patients with acute kala-azar were generally unreactive in each of these assays. Children with subclinical infection demonstrated relatively low levels of proliferation and interferon-gamma production, but none went on to develop overt kala-azar during the study. Patients evaluated after therapy for kala-azar demonstrated yet higher levels of lymphoproliferation and cytokine generation and produced low but significant levels of cytokines in vitro in response to parasite antigens, but not during the activation of infected macrophages. Finally, peripheral blood mononuclear cells from adults with positive delayed-type hypersensitivity responses and no history of kala-azar showed the broadest reactivity in vitro. These patients' cells generated the largest amounts of activating cytokines in vitro during the activation of autologous macrophages to a leishmanicidal state.
Peripheral blood eosinophil counts and serum levels and in vitro production of eosinophilopoietic cytokines were assessed before and at frequent intervals after diethylcarbamazine treatment of Bancroftian filariasis. Eosinophil counts peaked at day 7 after the start of treatment (359% +/- 118% of pretreatment levels) and declined to pretreatment levels by day 17. Serum interleukin (IL)-5, undetectable in 14 of 15 patients before treatment, rose sharply but transiently, with peak levels (32 +/- 7 pg/mL) 2 days after diethylcarbamazine treatment. Granulocyte-macrophage colony-stimulating factor and IL-3 were not detectable in serum at any time. In vitro mitogen-induced IL-5 levels decreased significantly in 7 of 9 patients 3 days after treatment when serum IL-5 was at near-peak levels. By day 10 IL-5 values increased in 8 of 9 patients compared with treatment values (P < .02). These data define the temporal relation between serum IL-5 levels and the subsequent development of eosinophilia and suggest that lymphocytes are the source of IL-5.
The objective of this phase IB trial was to determine if cyclophosphamide (CY) could enhance the immune effects of interleukin-2 (IL-2), and if it could, was there an optimal immunomodulatory dosage. IL-2 alone at 30 million IU/m2 thrice weekly for 6 weeks or in combination with varying dosages of CY (300, 600, and 1,200 mg/m2) administered 3 days before IL-2 and repeated 3 weeks later was given to consecutive cohorts of patients (at least five per group) with advanced malignancies of varying types. To gauge the immune effects of the treatment, the variation in the amount of lymphokine-activated killer (LAK) cells generated in the peripheral blood mononuclear cells of patients and in a control group of normal volunteers was measured weekly over 7 consecutive weeks. Other immune parameters [natural killer cells (NK), peripheral blood eosinophils and lymphocytes, cell surface markers, soluble IL-2 receptor, and IL-2 antibodies] were also closely followed to study if they correlated with the degree of LAK activity. The group of patients that received low-dosage CY (300 mg/m2) and IL-2 produced the highest and most sustained levels of LAK and NK activity (p < 0.05) when compared with the cohorts receiving IL-2 alone or to those receiving the higher dosages of CY. No other immune parameter showed a significant difference between the groups. Although low-dosage CY does increase the LAK activity seen with IL-2, only randomized clinical trials can determine if this enhancement will improve tumor responses.
To gain insight into the functional capacity of human T cells in the immune response against Mycobacterium tuberculosis, we evaluated the spectrum of cytokines produced by mycobacterium-reactive human T-cell clones. Nine of 11 T-cell clones bearing alpha beta or gamma delta T-cell receptors produced both Th1 and Th2 cytokines, a pattern resembling that of murine Th0 clones. The most frequent pattern was secretion of gamma interferon, tumor necrosis factor alpha (TNF), and interleukin-10 (IL-10), in combination with IL-2, IL-5, or both. Two clones produced only Th1 cytokines, and none produced exclusively Th2 cytokines. Although IL-4 was not detected in cell culture supernatants, IL-4 mRNA was detected by polymerase chain reaction amplification in two of six clones. There were no differences between the cytokine profiles of alpha beta and gamma delta T cells. A striking finding was the markedly elevated concentrations of TNF in clone supernatants, independent of the other cytokines produced. Supernatants from mycobacterium-stimulated T-cell clones, in combination with granulocyte-macrophage colony-stimulating factor, induced aggregation of bone-marrow-derived macrophages, and this effect was abrogated by antibodies to TNF. The addition of recombinant TNF to granulocyte-macrophage colony-stimulating factor markedly enhanced macrophage aggregation, indicating that TNF produced by T cells may be an important costimulus for the granulomatous host response to mycobacteria. The cytokines produced by T cells may exert immunoregulatory and immunopathologic effects and thus mediate some of the clinical manifestations of tuberculosis.
Levels of cytokine mRNA were studied in the central nervous system (CNS) of SCID mice infected with Toxoplasma gondii. This infection led to 100% mortality by day 23 postinfection. Inflammation was observed in the lungs on day 7 and in the heart, liver, and kidneys on days 14 and 18 of infection. In the CNS, necrotic, acellular lesions that contained numerous parasites, accompanied by a localized astrocyte activation, were evident on day 14. Polymerase chain reaction-assisted amplification of RNA revealed that, although transcripts for interleukin-1 alpha (IL-1 alpha) and IL-1 beta were present in the brains of uninfected mice, increased levels of these transcripts were detected on day 7 of infection. Transcripts for macrophage inflammatory protein 1 and transforming growth factor beta were also detected in brains of infected mice at this time point. On days 14 and 18, levels of these transcripts had increased and transcripts for IL-6, IL-10, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were also detected. Transcripts for IL-2 or IL-4 were not detected at any of the time points. Detection of locally produced cytokine transcripts may reflect involvement of the cytokines in the immunopathogenesis of this infection or involvement in mediating antitoxoplasma activity. To assess the possible role of endogenous IFN-gamma, TNF-alpha, IL-10, IL-6, and GM-CSF, cytokine-neutralizing monoclonal antibodies were administered to infected SCID mice. Neutralization of IFN-gamma or TNF-alpha led to earlier mortality than that in controls. In contrast, treatment with antibody to IL-10 and IL-6 increased survival time. Treatment with anti-GM-CSF did not alter the time to death. These results indicate that TNF-alpha and IFN-gamma are both involved in T-cell-independent mechanisms of resistance to T. gondii in SCID mice and that IL-10 and IL-6 may downregulate the immune response to this pathogen.
Clinical and immunologic evidence suggests that tuberculous pleuritis provides a model to understand protective immune mechanisms against Mycobacterium tuberculosis. We therefore evaluated the pattern of cytokine mRNA expression and cytokine production in pleural fluid and blood of patients with tuberculous pleuritis. RNA was extracted from mononuclear cells, reverse transcribed to cDNA, and amplified by polymerase chain reaction (PCR). After normalization for T-cell cDNA, cDNA from pleural fluid cells and peripheral blood mononuclear cells (PBMC) was amplified with cytokine-specific primers. PCR product was quantified by Southern blot. For the Th1 cytokines gamma interferon (IFN-gamma) and interleukin-2 (IL-2), PCR product was greater in pleural fluid than in blood, whereas PCR product for the Th2 cytokine IL-4 was decreased in pleural fluid compared with blood. Concentrations of IFN-gamma were elevated in pleural fluid compared with serum, but IL-2, IL-4, and IL-5 were not detectable. Mean concentrations of IFN-gamma and IL-2 in supernatants of M. tuberculosis-stimulated pleural fluid cells were significantly greater than corresponding concentrations in supernatants of stimulated PBMC. In situ hybridization showed that increased IFN-gamma production by pleural fluid cells was associated with a 20- to 60-fold increase in the frequency of antigen-reactive IFN-gamma-mRNA-expressing cells. Because IL-10 can be produced by T cells and macrophages, pleural fluid cells and PBMC were normalized for beta-actin cDNA content and then amplified by PCR with IL-10-specific primers. IL-10 mRNA was greater in pleural fluid cells than in PBMC and was expressed predominantly by macrophages. IL-10 concentrations were elevated in pleural fluid versus serum. These data provide strong evidence for compartmentalization of Th1 cytokines and IL-10 at the site of disease in humans with a resistant immune response to mycobacterial infection.
The mechanism of airway eosinophilia during antigen-induced inflammation was investigated by measurement of eosinophil-active cytokines utilizing an eosinophil survival assay. In the first study, 4 patients with allergic rhinitis underwent segmental bronchoprovocation (SBP) with low, medium, and high doses of ragweed extract instilled into different bronchial subsegments; bronchoalveolar lavage (BAL) fluids were collected from each segment 12 min and 48 h after challenge. Eosinophil granule proteins and eosinophil survival activity were significantly elevated in the 48-h (late-phase) BAL fluids from these segments. Correlations were observed between the concentrations of eosinophil granule proteins and eosinophil survival activity (rs = 0.717 to 0.880, p < 0.001) in BAL fluids. Eosinophil survival activity was completely neutralized by anti-IL-5 monoclonal antibody in five of the seven 48-h samples tested representing three of the 4 patients. In the two remaining samples, eosinophil survival activity was only partially neutralized by either anti-IL-5 antibody or anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) but was completely neutralized by anti-IL-5 and anti-GM-CSF in combination. Subsequently, in the second study, 10 patients with allergic rhinitis were challenged by SBP with ragweed extract. Eosinophil survival activity was significantly elevated in the 48-h BAL fluids; this activity was partially neutralized by anti-IL-5 antibody about (48%) and completely neutralized by the combination of anti-IL-5 and anti-GM-CSF antibodies. These findings suggest that the eosinophil survival activity in the late inflammatory lesions following SBP with allergen is mainly associated with IL-5, with small contributions from GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)
The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.
Patients with acute kala azar are generally nonreactive in a number of immunologic assays, including T cell proliferation and generation of macrophage-activating cytokines, principally IFN-gamma, in response to leishmania antigens in vitro. To test for potential immunosuppressive factors, a series of T cell lines and clones were established from patients with acute kala azar, from patients after chemotherapy for kala azar, and from skin test-positive adults from the same endemic region. Although CD4+ T cell lines and clones could be readily established from the skin test-positive adults, lines and clones from acute or treated patients were heavily biased in expression of CD8+. The CD8+ cells from acute patients did not themselves release cytokines in response to leishmania antigens in vitro, but markedly affected the cytokine profile of peripheral blood mononuclear cells isolated 1 yr later after recovery. Addition of the CD8+ cells caused inhibition of lymphoproliferation and IFN-gamma release, with augmentation of IL-6 and IL-10 release. The inhibitory effects of the CD8+ cells could be partially abrogated by antibodies to IL-10 but not by antibodies to IL-4. Analysis of four patients with acute kala azar demonstrated release of IL-10 that could not be demonstrated in supernatants from asymptomatic skin test-positive individuals. Generation of IL-10 may contribute to the profound suppression of IFN-gamma release that occurs during kala azar due to Leishmania chagasi.
Administration of lipopolysaccharide (LPS) results in activation of the hypothalamic-pituitary-adrenal axis. LPS induces the release of a number of proinflammatory cytokines, i.e. interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF), which activate the hypothalamic-pituitary-adrenal axis as well and may mediate the effects of LPS. Variations in the kinetics of appearance of IL-1, TNF, and IL-6 after LPS challenge suggested that these cytokines may play different roles at different times. To elucidate the mutual dependence and contribution of individual cytokines in the course of LPS-induced ACTH release, we used blocking antibodies to IL-6, TNF, and the IL-1 receptor. Our results demonstrate that anti-IL-6 antibody abrogated ACTH induction throughout the course of the response both 2 and 4 h after LPS challenge. In contrast, anti-IL-1 receptor and anti-TNF antibody, given individually, blocked ACTH production at 4 h, but not at 2 h. Only combined administration of these two antibodies diminished, but did not eliminate, ACTH release at 2 h. This is the first demonstration that all three inflammatory cytokines are obligatory for LPS-induced elevation of plasma ACTH. In addition, these results suggest that IL-1, IL-6, and TNF play different roles in LPS-induced ACTH release.