Thomas R. Ulich

Harvesting the Noncirculating Pool of Polymorphonuclear Leukocytes in Rats by Hetastarch Exchange Transfusion (HET): Yield and Functional Assessment

Isolation of polymorphonuclear leukocytes (PMN) provides an opportunity to study PMN activity in vitro and to label PMN for study of in vivo kinetics. However, simple phlebotomy (SP) of a small animal frequently yields too few PMN for in vitro handling, while PMN harvested from an induced‐peritonitis may not accurately reflect PMN in a less stimulated state. We report a novel method of harverting PMN from the circulation of rats, using hetastarch exchange transfusion (HET), which is both time and animal sparing. HET harvested 8‐fold more PMN than SP. In vitro cell function was examined with assays of adherence, chemotaxis, bacterial killing, and superoxide generation. No significant (p < 0.05) difference was found between PMN obtained by HET and pooled‐PMN obtained by SP. In vivo function was examined following labeling with indium111‐oxine. The kinetics pattern described suggested normal migratory activity when compared to previous reports. The data demonstrate that rats possess a relatively large, noncirculating pool of PMN which is readily accessible by HET.

Mechanisms of tumor necrosis factor alpha-induced lymphopenia, neutropenia, and biphasic neutrophilia: a study of lymphocyte recirculation and hematologic interactions of TNF alpha with endogenous mediators of leukocyte trafficking.

Tumor necrosis factor alpha (TNF) induces lymphopenia, neutropenia, and biphasic neutrophilia after intravenous injection of 3,000 U TNF in Lewis rats. The mechanism of TNF‐induced lymphopenia was investigated by means of thoracic duct cannulation. Hourly measurements of lymphocyte recirculation via the thoracic duct failed to reveal any significant decrease in lymphocyte recirculation in TNF‐treated vs. control rats, suggesting that a decrease in lymphocyte recirculation through the thoracic duct is not the mechanism for TNF‐induced lymphopenia. The mechanism of TNF‐induced neutropenia was investigated by administering TNF to rats in whom a neutrophilia had been induced with Interleukin‐1 (IL‐1). In rats with neutrophilia, TNF resulted in a sharp decrease in the circulating neutrophil pool, demonstrating that TNF induces neutropenia by causing neutrophils to leave the circulating pool rather than decreasing neutrophil release from the marrow. The mechanism of neutropenia was furthermore shown to be due to the transient intravascular margination of neutrophils by administering epinephrine concomitantly with TNF. Epinephrine, which causes neutrophilia solely by demargination, abrogated the TNF‐induced neutropenia and actually resulted in a neutrophilia that was greater than the neutrophilia occurring in epinephrine alone‐treated rats, demonstrating both that TNF had already caused release of marrow neutrophils at the time of peripheral neutropenia, and that the paradoxical neutropenia was due to the transient intravascular margination of neutrophils. The known property of epinephrine to cause neutrophilia exclusively by demargination was proved by examination of the bone marrow of epinephrine‐treated rats in whom no decrease in marrow neutrophils was observed (in contrast to TNF‐ and IL‐1‐treated rats in whom neutrophilia is accompanied by a depletion of marrow neutrophils). The mechanism of TNF‐induced neutrophilia was investigated by modulating the magnitude of both the first and second peaks of neutrophilia by priming of rats with daily injections of IFNγ for 2 days prior to administration of TNF. The first peak of neutrophilia in IFNγ‐primed TNF‐treated rats was decreased in comparison to TNF alone‐treated rats because of the well‐known neutropenic and myelosuppressive effect of IFNγ, which resulted in a decrease in the number of neutrophils that could be recruited to cause neutrophilia. The second peak of neutrophilia in IFNγ‐primed TNF‐treated rats, however, was increased in relation to the magnitude of the first peak, an observation that is consistent with the known priming effect of IFNγ on macrophages and the concept that the second peak of neutrophilia is due to the release of endogenous monokines. A possible negative feedback role for TNF in contributing to the in vivo regulation of biological responsiveness to TNF itself was tested by administering TNF to C3H/HeJ mice (which are deficient in TNF production but not TNF receptors) and to congenic C3H/HeN mice (which are not deficient in TNF). TNF induced a greater neutropenia and a greater neutrophilia in C3H/HeJ than in C3H/HeN mice, suggesting that a tonic deficiency in TNF results in a hyperresponsive state that might be postulated to represent upregulation of TNF receptors. In conclusion, experimental evidence is presented consistent with the hypotheses that TNF‐induced lymphopenia and neutropenia are due to transient intravascular margination, that the second peak of TNF‐induced neutrophilia is due to the release of endogenous monokines, and that the state of neutrophil responsiveness to TNF may be regulated by TNF itself by a negative feedback mechanism.

Purified native and recombinant human alpha lymphotoxin [tumor necrosis factor (TNF)-beta] induces inflammatory reactions in normal skin

These studies report findings that demonstrate that human alpha lymphotoxin (LT) induces local, visible, and microscopic inflammatory reactions in normal skin. Skin sites in rabbits, when inoculated with a single injection of native or recombinant human alpha lymphotoxin, demonstrated erythema, swelling, and warmth within 5 hr. Erythema peaked between 24 and 48 hr and resolved by 72 hr. Histologic studies of skin sites injected with native LT revealed polymorphonuclear neutrophil (PMN) infiltration and edema beginning as early as 3 hr posttreatment. Individual skin sites that received three daily injections of native LT exhibited persistent erythema and swelling. Palpable induration was evident 24 hr after the second injection in the series. Histologic examination revealed the presence of many PMNs with associated focal dermal destruction, in the form of microabscesses, and scattered mononuclear cells. In contrast, control materials and recombinant human tumor necrosis factor (TNF-alpha) did not induce visible skin reactions in the rabbit. Several additional controls excluded endotoxin as being the agent responsible for the inflammatory skin reactions observed. The ability of LT to induce inflammation may have a role in its antitumor activity and it may be an important endogenous mediator in other immunologic reactions.

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the neutrophilic infiltrate of experimental tubulointerstitial nephritis in the Brown-Norway rat by decomplementation

Anti-tubular basement membrane (TBM) antibody-associated tubulointerstitial nephritis (TIN) in Brown-Norway rats is induced by immunization with bovine TBM antigens and adjuvants. The lesion is characterized by linear deposition of IgG and C3 along the TBM with sequential neutrophil (Days 8-9)- and mononuclear (Day 10 and after)-dominated inflammatory infiltrates. To study the complement dependence of the infiltrative process, immunized rats were decomplemented with cobra venom factor (CVF). The CVF treatment did not affect the production or renal deposition of anti-TBM antibodies. CVF markedly reduced the neutrophilic inflammatory infiltrate. In rats immunized with suboptimal doses of soluble bovine TBM antigens to produce a mild lesion, decomplementation also decreased the mononuclear inflammatory infiltrates on Days 10-13. In rats immunized optimally with particulate TBM to induce maximally severe TIN, decomplementation did not affect the mild mononuclear cell infiltrate on Days 8 and 9 but did somewhat reduce the subsequent mononuclear infiltrate on Days 10 and 12. These results demonstrate that the anti-TBM antibody- and C3-associated neutrophilic inflammatory infiltrate is largely complement dependent. The early mononuclear cell infiltrate that was unmodified by CVF treatment may be dependent on complement-independent humoral events or related to cell-mediated immune events. A portion of the later mononuclear inflammatory infiltrate could be dependent on the preceding neutrophilic inflammatory phase.

The Effects of a Stable Analogue of PGE1 on the IgG Subclass Response to Particulate Bovine Tubular Basement Membrane in the Brown-Norway Rat

Abstract The IgG subclass and the IgM isotype response to immunization with particulate bovine tubular basement membrane (TBM) and adjuvants was studied in Brown-Norway rats receiving daily injections of a stable analogue of PGE1 (M-PGE1). M-PGE1 slightly reduced the average quantity of circulating TBM antibody as well as the average quantity of eluted IgG per gram of renal tissue as compared to controls. However, M-PGE1 did not qualitatively affect the distribution of the IgG subclass or IgM isotype response to TBM. The IgG response, which occurred predominantly in the IgG1 and IgG2a subclasses, increased from Days 8 to 14 after immunization, while the IgM response decreased over the same time period. The percentage of TBM antibody in the IgG2b subclass was markedly decreased as compared to the percentage of IgG2b antibody in total IgG. A substantial heterogeneity in the IgG subclass response was noted among individual rats with IgG1 constituting from 46 to 82% of circulating TBM antibody. Although no correlation between the IgG subclass response and the severity of tubulointerstitial nephritis was noted, heterogeneity in the IgG subclass response to autoantigens may, nevertheless, theoretically play an important role in the pathogenesis of autoimmune inflammatory phenomena.