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J Grooten

Publications and source records attributed to J Grooten.

53 records · Page 3Linked to original sources
PubMed1993

Increased IL-6 production and IL-6-mediated Ig secretion in murine host-vs-graft disease.

BALB/c mice neonatally injected with semiallogenic (A/J x BALB/c)F1 splenocytes develop a host-vs-graft (HVG) reaction between host T cells and donor B cells, resulting in hypergammaglobulinemia, splenomegaly, and increased serum levels of various autoantibodies. This syndrome is associated with a polyclonal activation of the donor-derived B cells. High serum levels of IL-6 were found in 4-wk-old mice undergoing HVG disease (mean +/- SEM, 132 +/- 93 as compared with 12 +/- 2 in control mice, p < 0.05). Also supernatants of spleen cell cultures from HVG mice contained increased levels of IL-6. In situ hybridization and cell depletion experiments demonstrated that host macrophages were responsible for this pathologic IL-6 secretion. The spontaneous in vitro production of autoreactive antibodies by donor B cells from HVG mice was further enhanced by adding human rIL-6, whereas addition of human rIL-1 beta, human rIL-2, murine rIL-4, murine rIL-5, or combinations of these cytokines had no effect. Finally, addition of blocking anti-IL-6 and anti-IL-6 receptor mAb markedly reduced hyper IgG1 production in cultures of spleen cells from HVG mice. These data suggest that an increased production of IL-6 by persistently stimulated host macrophages is involved in the activation of donor B cells leading to HVG disease.

Animals↗
PubMed1993

Cell membrane permeabilization and cellular collapse, followed by loss of dehydrogenase activity: early events in tumour necrosis factor-induced cytotoxicity.

Early events in the cytotoxic response to tumour necrosis factor (TNF) of the murine fibrosarcoma cell lines L929 and WEHI164cl13 were assessed on a cell by cell basis using the fluorescent exclusion dye propidium iodide (PI) and analysis by flow cytometry. The rationale of this approach is based on the exclusion of PI by cells with intact membranes. PI-positive cells appeared within a few hours of TNF treatment and further accumulated with time at a TNF dose-dependent rate. Thus, TNF rapidly caused a breakdown of the barrier function of the membrane in these TNF-sensitive fibrosarcoma cell lines. On a time basis, membrane permeabilization was immediately followed by a sudden shrinkage of the cell and was accompanied by cell death, but preceded the inactivation of the mitochondrial succinate dehydrogenase by several hours. The latter enzymatic activity was measured by the MTT chromogenic assay. Cell death was determined on the basis of the capability of individual cells to produce a progeny in a clonogenicity assay. Both membrane permeabilization and cellular collapse were fast events that were completed within a very short time and may represent the direct cause for cell death. Opposed to this, loss of mitochondrial succinate dehydrogenase activity evolved more slowly, was initiated at a later time and apparently represents a post-lethal event, not directly linked to the TNF signal transduction pathway. Finally, the enhancing effect of the protein synthesis inhibitor cycloheximide on the various features of TNF-induced cytotoxicity was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effect of bcl-2 proto-oncogene expression on cellular sensitivity to tumor necrosis factor-mediated cytotoxicity.

Introduction and expression of the proto-oncogene bcl-2 (B-cell lymphoma/leukemia 2) has been shown to extend the survival of certain hematopoietic cell lines after growth factor deprivation, by blocking apoptosis or programmed cell death. We investigated the effect of bcl-2 expression on cellular sensitivity to lysis by tumor necrosis factor (TNF), a cytokine capable of inducing apoptosis in several tumor cell lines. Introduction of the human bcl-2 gene in the highly TNF-sensitive L929 mouse fibrosarcoma cell line did not result in altered TNF sensitivity. Likewise, NIH3T3 and REF cells, which are resistant to TNF cytotoxicity but become TNF sensitive upon cotreatment with actinomycin D or upon expression of the adenovirus E1A gene, did not show altered TNF sensitivity upon bcl-2 transfection. Despite constitutive expression of the endogenous bcl-2 gene, human MCF7 breast carcinoma cells, as well as HL60 promyelocytic leukemia and U937 histiocytic lymphoma cell lines were found to be TNF sensitive. bcl-2-overexpressing derivatives of these cell lines did not acquire reduced TNF sensitivity and still exhibited the characteristic pattern of internucleosomal DNA fragmentation of TNF-induced apoptosis. Moreover, bcl-2 expression in the interleukin 3 (IL-3)-dependent myeloid cell line 32D protected these cells from apoptosis resulting from growth factor deprivation, but not from apoptosis induced by TNF. These data clearly establish the absence of a correlation between bcl-2 gene expression and cellular sensitivity to TNF-induced cell lysis. These findings are discussed in the context of the hypothesis of different pathways for induction of apoptosis, only some of which are affected by bcl-2 expression.

3T3 Cells↗
PubMed1992

Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma.

We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

Animals↗
PubMed1991

Expression of the tumor necrosis factor gene in tumor cells correlates with reduced tumorigenicity and reduced invasiveness in vivo.

We investigated whether a constitutive production of low amounts of tumor necrosis factor (TNF) by neoplastic cells affects their in vivo tumorigenicity. TNF-resistant derivatives were isolated from the TNF-sensitive murine fibrosarcoma cell lines L929s and WEHI164cl13s, L929r1-type TNF-resistant subclones were found to constitutively produce TNF in vitro, in contrast to non-TNF-producing but TNF-resistant L929r2 and WEHI164cl13r2 cell clones. The TNF-sensitive parental cell lines as well as the L929r2 and WEHI164cl13r2 cell lines similarly induced fast-growing tumors upon s.c. inoculation into nude mice (Swiss-nu/nu). In contrast, the TNF-producing L929r1-type cells showed reduced tumorigenicity and in vivo growth rate, which both inversely correlated with the level of in vitro-produced TNF. Tumor take incidence but not the in vivo growth rate of L929r1-type cells was greatly facilitated by prior whole body gamma-irradiation (350 rads) of the recipient, implying the involvement of host mechanisms at least in the lower take incidence of L929r1 tumors. These host mechanisms, possibly activated by tumor-produced TNF, acted only locally, inasmuch as the growth of an inoculum of L929s cells was not influenced either by a simultaneous distant inoculum of L929r1 cells or by established, distant L929r1 tumors. Efforts to eliminate these host mechanisms by prior local UV irradiation of the skin were unsuccessful. All L929 cell types were found to be similarly susceptible to killing by host cytotoxic effector cells (macrophages and natural and lymphokine-activated killer cells). Histological investigation did not reveal clear differences in tumor-associated inflammatory cells but revealed that tumors induced by L929r1-type cells, in contrast to L929s and L929r2 tumors, did not show invasiveness in host tissues. Moreover, L929r1 tumors were frequently encapsulated, which was never observed for tumors induced by L929s and L929r2 cells. Taken together, our results suggest that tumor-derived TNF locally activates host antitumor activities. Possible effector mechanisms are discussed.

Animals↗
PubMed1990

IL-4 acts synergistically on the IL-2 response of an autoreactive T-cell clone; synergism correlates with increased intracellular IL-2, but not with a modified IL-2 receptor expression.

TE44, an H-2b-restricted, self-reactive T-cell line, did not produce autocrine-acting growth factors, neither after antigenic nor after mitogenic activation; they remained for their proliferation completely dependent on exogenously added IL-2. Administration of IL-4, which poorly promotes growth by itself, resulted in a 5- to 10-fold enhancement of the specific biological activity of IL-2 on antigen-activated TE44-cells. This synergism was exerted nonreciprocally and required the presence of both lymphokines. IL-4 did not affect the number, nor the affinity, nor the rate of internalization of the high-affinity receptors for IL-2. However, increased levels of intracellular IL-2 were observed, suggesting an effect of IL-4 on the turnover of IL-2. This might allow a prolonged activity of IL-2 or IL-2-associated molecules inside the cell. Furthermore, the lack of autocrine growth factor production by antigen-stimulated TE44 is discussed in terms of its relationship to the autoimmune specificity of these T-cells.

Animals↗
PubMed1989

An ongoing in vivo immune response affects the abundancy and differentiation of lymphokine-activated killer cell precursors, but does not influence their broad spectrum target reactivity.

Using a model of local lymph node (LN) immunization, we investigated the effect of in vivo Ir on the generation of lymphokine-activated killer (LAK) cells or their precursors. Ag used for immunization were SRBC, horse RBC, OVA, keyhole limpet hemocyanin, or CFA. Ag-draining LN, in the acute phase of the Ir, did not contain detectable LAK effector activity, nor an enhanced NK activity. After culture for 3 to 5 days in the absence of exogenously added IL-2, immunized LN cells developed a spontaneous LAK-like cytotoxicity. This activity represented a substantial fraction of the IL-2-generated LAK cytolysis and was mediated by a Thy-1+ cell population phenotypically indistinct from IL-2-induced LAK. Inclusion (on day 0 of culture) of antibodies to IL-2, IL-2R, IL-4, IL-6, IFN-gamma, or TNF suggests a marginal involvement of IL-2 and IL-4 in the generation of this response. LAK, induced in vitro by exogenously added IL-2, developed earlier in LN cells immunized with particle Ag (SRBC, horse RBC, and CFA), but not with protein Ag (OVA and keyhole limpet hemocyanin). This effect was not mediated by endogenous IL-4. During further culture time in the presence of a saturating IL-2 concentration, similar levels of LAK activity were generated in naive and immunized LN cells. This agrees with the similar or slightly higher LAK precursor frequencies in immunized versus naive LN as assessed by limiting dilution experiments. Considering the 2.7-fold to 18-fold increase in cell content of the immunized LN, due to a recruitment and expansion of Ag-reactive B and T lymphocytes, a de novo generation of LAK precursors at the site of the Ir, and resulting from the Ir, must be assumed. In conclusion, our results suggest an interrelation between immune reactivity and LAK responses.

Animals↗
PubMed1989

Acquisition by the murine host of responsiveness toward various neoplastic cell lines, but not toward self, through adoptive transfer of a helper T-lymphocyte clone with antiself specificity.

Self-antigens, when expressed on neoplastic cells, have been shown to exhibit a certain antigenicity. We attempted to apply this antigenicity to enhance antitumor immune responses. Cells from the syngeneic, CD4+, CD8-, helper T-lymphocyte clone TE2 were adoptively transferred to C57BL/6 mice. TE2 lymphocytes recognize a self-antigen on splenocytes that is expressed aberrantly on the neoplastic cell lines EL4/8, EL4/13, B16-BL6, and PG19, all of C57BL/6 origin. Their adoptive transfer led to the rejection by the host of the former neoplastic cells and of 3LL carcinoma cells, administered 2 months later; inocula 40 to 80 times the minimal lethal size were rejected and conveyed to the mice a 10-fold enhanced cytotoxic T-lymphocyte response. Despite the autoimmune responsiveness of the TE2 T-lymphocytes, no graft-versus-host reaction was apparent. This conclusion is based on the absence of a polyclonal B-lymphocyte stimulation in the host, the stable number of residual donor TE2 cells, and the general health of the recipient mice. Consequently, the autoimmune and tumor-responsive TE2 cells, transplanted into the immune environment of the host, exhibit a specificity that is restricted toward neoplastic cells.

Animals↗
PubMed1987

Specific suppression elicited by EL4 lymphoma cells in syngeneic mice. Specificity includes self-antigens on EL4.

In vivo, subclones derived from EL4 lymphoma cells generate suppressor T lymphocytes specific for anti-EL4 immune responses. Spleen cells of EL4-sensitized C57BL/6 mice down-regulate the in vitro induction of EL4-specific cytolytic T lymphocytes (CTL). In addition, EL4-sensitized spleen cells interfere with the antigen response of two T lymphocyte clones. These recognize, in an H-2b context, a self-antigen on spleen cells that is also expressed by transformed cells, including EL4. The simultaneous anti-self and anti-EL4 specificity of the helper and suppressor activities suggests, therefore, that they are the product of an in vivo autoimmune reaction to EL4. The anti-self suppression might aim to re-establish self-tolerance, at the same time down-regulating responses against immunogenic epitopes that are co-expressed with the self-antigen on the EL4 cells. This agrees well with our observation that suppressor T cells, apparently elicited by suppressogenic epitopes on non-immunogenic EL4 subclones, down-regulate the CTL response elicited by immunogenic EL4 subclones. The additional self-specificity of this suppression indicates that the suppressogenic epitopes at least in part represent EL4 self-antigens.

Animals↗
PubMed1987

Alloinduced suppression and cytotoxicity: two functions of a single cell.

Using different experimental approaches we here show that the suppression and cytotoxicity generated during a one-way mixed lymphocyte culture (MLC) are mediated by a single cell population, in this case the cytotoxic T lymphocyte (CTL). Results of limiting-dilution analysis of cells from a 4-day MLC demonstrate the coexpression of both functions and argue against the existence of a separate suppressor cell population that regulates the in vitro alloresponse. The suppressive quality of CTL is also exemplified in cells with a clonal origin, such as cloned cytotoxic T lymphocytes and the mouse X rat hybridoma PC60. Further experiments demonstrate that this suppression is not primarily mediated through lysis of the stimulator cells, and studies in the PC60 model suggest that lysis and suppression may have different induction requirements.

Animals↗
PubMed1983

The immunomodulatory effect of anti-Micrococcus luteus antibodies. I. Effect on in vitro rabbit T cell functions.

A range of purified rabbit anti-Micrococcus luteus antibodies (anti-MCAb) were tested for their ability to interfere with a variety of in vitro immune responses. Such antibodies strongly inhibited the secondary IgG antibody response to sheep red blood cells without affecting the IgM response or the proliferative responses to mitogens and antigens. By exposing lymphocyte populations to anti-MCAb, it was found that such reagents exerted a strong mitogenic effect on rabbit T lymphocytes, provided these cells were derived from antigen-activated lymph nodes. This mitogenic effect was also obtained with F(ab')2 fragments of anti-MCAb and with hybridoma-derived anti-MCAb. Collectively, these data indicate that anti-MCAb inhibit the initiation of IgG synthesis possibly through the expansion of immunoregulatory T cell subsets.

Animals↗
PubMedDate not supplied

Modulation of the immune response by antibacterial antibodies.

Antibodies induced by the gram+ bacteria Micrococcus lysodeikticus exhibit different carbohydrate specificities and hence might cross-react with membrane glycoproteins and/or glycolipids of mammalian cells. Using a T cell derived lymphoid line, these antibodies were found to detect a membrane marker which is only exposed in confluent culture conditions on non-dividing cells. Such confluence related antigen (Cag) is a cryptic membrane antigen, which can be unmasked through membrane perturbating agents such as p-formaldehyde or through interactions with macrophages and macrophage derived factors. Anti-micrococcus antibodies appear also to affect functionally normal T lymphocytes. Thus such reagents drastically inhibit the murine T cell reactivity towards mitogens such as Con A and PHA provided, however, the mitogenic signal is delivered through peritoneal macrophages. Furthermore, anti-micrococcus antibodies induce activated T lymphocytes into mitogenesis, but not unprimed resting T cells. Hence the physiological activity of anti-micrococcus antibodies depends on the state of activation of the T lymphocyte, indicating the involvement of cryptic membrane molecules. The relevance of such phenomena to host-bacteria and host-parasite interaction will be discussed.

Animals↗
PubMedDate not supplied

TNF-induced intracellular signaling leading to gene induction or to cytotoxicity by necrosis or by apoptosis.

TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.

Animals↗