C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha.

We have demonstrated that purified C5a is a potent stimulus to human PBMC secretion of TNF-alpha, IL-1 beta, and IL-1 alpha, which proceeds in a dose-dependent fashion. At a given concentration of C5a, TNF-alpha and IL-1 beta secretion did not differ significantly; both were secreted in significantly greater quantity than IL-1 alpha. Clinical conditions such as Gram-positive and Gram-negative bacterial infections, trauma, and immune complex diseases activate complement. Through the mediation of TNF and IL-1 secreted in response to C5a, these diverse disorders can share common features of fever, coagulopathy, acute phase protein production, and disordered metabolism.

Hyperthermia and bleomycin schedules on V79 Chinese hamster cell cytotoxicity in vitro.

The effect of sequence and timing of hyperthermia (43 degrees) and bleomycin on Chinese hamster cells (V79) has been investigated. Hyperthermia preceding bleomycin treatment produced a greater cytotoxic effect than bleomycin treatment preceding hyperthermia. Furthermore, it appears that the combination of hyperthermia and bleomycin becomes less effective if the application of bleomycin is delayed. The enhancement of cytotoxicity with hyperthermia first may be related to the effect of heat on the intracellular bleomycin degradation ability and protein synthesis. V79 cells treated with a protein synthesis inhibitor, cycloheximide, before bleomycin (but not in the reversed sequence) also showed a markedly lower level of survival. As for hyperthermia treatment, pretreatment with cycloheximide did not change the uptake of bleomycin. These results suggest that hyperthermia and cycloheximide have increased the effectiveness of bleomycin and are consistent with the observation on chromosome damage induced by hyperthermia-bleomycin and cycloheximide-bleomycin treatments reported in the literature.

Effects of iron, copper, cobalt, and their chelators on the cytotoxicity of bleomycin.

Bleomycin is widely used for treating several types of human tumors as well as a variety of experimental tumors. The ability of this antibiotic to bind and to damage DNA has been proposed to be responsible for its antitumor effect. Bleomycin is also a good chelator for several metals, e.g., iron, copper, and others. Bleomycin:metal complexes have been investigated in detail particularly for their action on isolated DNA. The conclusions from these studies indicate that metal-chelated bleomycin either is ineffective or more effective in damaging DNA. In this paper, we tested the effect of iron, copper, cobalt, and their chelators on bleomycin cytotoxicity. Our results suggest that chelating bleomycin with copper or adding an iron chelator (deferoxamine), diethylenetriamine pentaacetic acid, and a copper chelator (penicillamine) shows no effect on bleomycin cytotoxicity. On the other hand, iron dextran and a metal chelator, diethyldithiocarbamate (DDC), with bleomycin show enhanced cytotoxicity. Cobalt-chelated bleomycin is not cytotoxic but is cytotoxic when combined with DDC. We suggest that different mechanisms are contributing to the enhanced toxicity of bleomycin with iron dextran and DDC. Bleomycin acts as a ferrous oxidase which promotes the iron toxicity. In the case of DDC, it can act as a reducing agent or it can help to maintain the bleomycin:metal complex in the reduced form which can generate radicals.

Modification of membrane function, protein synthesis, and heat killing effect in cultured Chinese hamster cells by glycerol and D2O1.

Glycerol and D2O can provide a protective effect to Chinese hamster V79 cells receiving heat treatment. The mechanism for this effect is assumed to be the ability of these agents to stabilize proteins which implies that one of the possible heat-killing mechanisms is the inactivation of a heat-sensitive molecule (protein?). In addition, we observed that heat can alter the membrane permeability rapidly, and glycerol can reduce the initial heat-induced membrane permeability changes (determined by aminoisobutyric acid uptake). Although not as effective as cycloheximide, glycerol and D2O can retard protein synthesis. These two processes can add to the protective effect of stabilizing cellular protein against heat killing. Since glycerol and D2O added during the heat-conditioning period can interfere with the appearance of thermotolerance, the mechanisms for the protective effect of glycerol and D2O are likely to be different from heat-induced thermotolerance. We propose that the heat-sensitive molecule protected by D2O or glycerol may also play a role in the triggering process of the thermotolerance phenomenon. If the conditioning heat treatment is sufficient to affect this molecule but not other cellular targets, thus allowing the cell to survive, thermotolerance may be observed in surviving cells in response to second or continued heat treatment. Depending on the severity of the heat effect, this heat-sensitive molecule may continue to exist after a conditioning heat treatment in medium containing glycerol or D2O, and therefore, the response of the cells to the challenging heat is altered little. This proposed mechanism is capable of explaining several thermotolerance experimental protocols. Since glycerol can also reduce the toxicity of vincristine, microtubule-related protein is probably one of the proteins stabilized by this agent. D2O also probably affects microtubule protein, because the cells heated in medium containing D2O show little topological changes.

Impairment of Na+-dependent amino acid transport in a cultured human T-cell line by hyperthermia and irradiation.

We have examined the effects of hyperthermia and radiation on the ability of a human T-leukemic lymphocyte line (Molt-4) to transport the Na+-dependent amino acid, 2-aminoisobutyrate (AIB). Heating Molt-4 at 43 degrees for 1 to 4 hr damages the ability of these cells to accumulate AIB. The damage to the transport system at 43 degrees impairs only the maximal rate of AIB uptake, i.e., Vmax. The thermal effect on AIB transport parallels the radiation effects observed for this system. Preliminary data indicate that heat and radiation may induce irreversible transitions in the tertiary or quaternary structure of a plasma membrane protein involved in regulating Na+-dependent amino acid transport. However, the mechanism by which heat and radiation damage this protein is different.

Involvement of sulfhydryl groups in the action of the insulin and radiation on thymocyte Na+-dependent amino acid transport.

1,p-Chloromercuribenzene sulfonate concentrations less than 10(-5) M stimulate the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations greater than 10(-5) M of this reagent, transport is impaired and cell viability is effected. In contrast, 5,5'-dithiobis-(2-nitrobenzoate) between 10(-4) and 10(-6) M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. Treatment of thymocytes with 10(-4)M 5,5'-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 - 10(7)-7 - 10(7)/cell, activates 2-aminoisobutyrate transport to the same extent as does p-chloromercuribenzene sulfonate. Cells treated with 10(-6) M insulin showed a 30-50% reduction in the number of sulfhydryl groups that could be titrated with 5,5'-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10(-6) M p-chloro(203Hg)mercuribenzene sulfonate, addition of 10(-6) or 10(-9) M insulin before treatment with the sulhydryl reagent again reduces the number of titrable SH groups by 20%. 3. Insulin (10(-10)-10(-6) M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not additive. 4. Insulin, but not p-chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by gamma-irradiation. Treatment of cells with p-chloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. gamma-irradiation reduces the number of 5,5'-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.

A comparison of the effects of hyperthermia on cell growth in human T and B lymphoid cells: relationship to alterations in plasma membrane transport properties.

Hyperthermic exposure (39-43 degrees C) for 1 or 2 hr impairs growth and Na+-dependent amino acid transport in both a radiosensitive human T (Molt-4) and a radioresistant B (RPMI 1788) lymphoid cell line. The heat damage to Na+-dependent amino acid transport in both cell lines is reversible under the conditions tested. Cell growth, as judged by increases in cell number, is decreased in both cell lines after hyperthermic treatment (43 degrees C, 1-hr exposure). This decrease in growth correlated with the damage to, and recovery of, the Na+-dependent amino acid transport system. However, the sensitivity to heat of both growth and Na+-dependent amino acid transport appears to differ in Molt-4 which is somewhat more sensitive to hyperthermia (T-cell line) vs RPMI-1788 (B-cell line). In the case of Molt-4, the rate of growth is decreased for about 60-80 hr after cells are exposed for 1 hr at 43 degrees C; whereas increases in cell number in the RPMI 1788 is observed within 40 hr after the heat treatment. The differences observed in cell growth and transport in these two lymphoid cell lines are attributed to the manner in which heat affects (i) the transport parameters in Molt-4 vs RPMI 1788 (i.e., the Michaelis-Menten constants Km and Vmax) and (ii) the putative plasma membrane sulfhydryl protein(s) which modulates Na+-dependent amino acid transport.

Protection of heat induced cytoxicity by glycerol.

Glycerol, at concentrations of 2-10% is a potent hyperthermic (43 degrees - 45 degrees C)protector of cultured Chinese hamster cells, V79. Furthermore, the sensitization effect of low pH on heat death is also drastically reduced by the addition of glycerol into the culture medium. Together with the known cellular effects of heat and the role of glycerol in various cellular structures and functions, the data suggest that microtubules and membranes may be involved in the expression of heat-induced cell death.

An in vitro model of the wound microenvironment: local phagocytic cell abnormalities associated with in situ complement activation.

An in vitro model was developed to investigate the inflammatory response to tissue damage. Human fibroblasts were heat killed and incubated with serum. Complement studies showed activation of the alternative pathway proportional to the number of dead cells; C3 was fixed on dead cells, and C5a was generated. Neutrophils (PMNLs) adhered to killed fibroblasts, a process requiring fresh serum. After adhering to killed fibroblasts in the presence of serum, PMNLs exhibited depressed chemotactic responsiveness to activated serum and reduced bactericidal activity against preopsonized Staphylococcus aureus. These data suggest that thermally killed cells activate and fix complement, a process generating cleavage products that, in turn, recruit PMNLs and bind them to the inflammatory site. Thus, in our model, dead tissue activates humoral mechanisms and inflammatory cells; this process results in depressed in situ host-defense function upon subsequent local challenge with microbes.

Modification of rat thymocyte membrane properties by hyperthermia and ionizing radiation.

Thymocytes are one the most widely used cell models for the study of radiation-induced interphase death. This cell-type was chosen for the study of hyperthermic and radiation effects on two membrane-related processes implicated in the interphase death of cells: Na+-dependent 2-aminoisobutyric acid (AIB) transport and cyclic 3'-5' adenosine monophsophate formation. The response of AIB transport to heat is dose-dependent, but the biphasic thermal response curve (AIB uptake versus time) differs fom the sigmoidal radiation response curve. Heating thymocytes for 20-30 min at 43 degrees C stimulates AIB uptake. Additional heating at 43 degrees C, however, markedly reduces AIB uptake. Despite the immediate stimulating effect of heat (30 min at 43 degrees C), the thymocyte has already developed irrepairable impairments, as demonstrated by the fractionated heating experiments. The heat-induced impairment of AIB uptake is mainly on the Na+-dependent component of neutral amino-acid transport, affecting primarily the maximal rate of uptake, i.e. Vmax. Additional evidence for heat-induced plasma membrane damage is the alteration in cAMP levels. Heating thymocytes for 30 min or longer at 43 degrees C causes a massive rise in cAMP level within the cell. This differs from thymocytes exposed to radiation where no rise in cAMP is observed.