Failure of rabbit neutrophils to secrete endogenous pyrogen when stimulated with staphylococci.

Cells obtained from acute peritoneal exudates in rabbits were separated into neutrophil and mononuclear populations by centrifugation on colloidal silica gradients. When these populations were separately incubated in tissue culture medium in the presence of opsonized Staphylococcus epidermidis, endogenous pyrogen was secreted only by the adherent cells of the mononuclear population. Pyrogen production by neutrophils could not have amounted to as much as 1% of the pyrogen produced by macrophages. When mononuclear cells were added back to purified neutrophils, no pyrogen was produced that could not be accounted for by the number of macrophages added. Rabbit blood cells were similarly fractionated on colloidal silica gradients. Again, endogenous pyrogen was made only by the adherent mononuclear population. The neutrophils isolated on these gradients appeared to be morphologically normal and were 85% viable as judged by dye exclusion. They showed normal random motility. Both blood and exudate neutrophils responded chemotactically to N-formyl Met-Leu-Phe, and blood neutrophils responded chemotactically to zymosan-activated serum. Both kinds of neutrophils phagocytosed zymosan particles and both killed opsonized S. epidermidis in a roller tube system. Both blood and exudate neutrophils showed normal superoxide production when stimulated with opsonized zymosan particles. This evidence suggests that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that they contain.

Fever, temperature, and the immune response.

Fever's ability to manipulate the character and extent of physiological temperature gradients correlates with the unusual influence different physiological temperatures have upon model immune responses in vitro. This relationship may help to explain the remarkable evolutionary conservation of the febrile response to infection. A very restricted range of the upper physiological temperatures supports the activation of resting lymphocytes for proliferation and effector formation in the two major limbs of the immune system, cell-mediated immunity and humoral immunity. In contrast, once effectors are formed they can function in a fashion which is nearly independent of physiological temperature. This suggests that physiological temperature change acts to regulate the emergence of new immune responses but does not restrict the activity of existing effector mechanisms once they have been formed. The differential sensitivity of these processes to different physiological temperatures suggests that fever's biological purpose with respect to the immune system is the elimination of lower peripheral tissue temperatures rather than the elevation of core temperatures. However, further studies may reveal that some functions are amplified by the core temperature transitions while other functions are selectively regulated by peripheral tissue temperature transitions. The critical cell for the temperature dependence of immune responses seems to be the Th since its ability to produce cytokines is highly temperature dependent. In contrast, macrophages produce cytokines equally well at all temperatures except those of the febrile core, a feature which may serve to downregulate the production of endogenous pyrogens.

Oral infectivity and bacterial interactions with mononuclear phagocytes.

The purpose of this study was to clarify the association between the oral infectivity of a bacterial strain and its susceptibility to ingestion by mononuclear phagocytes or ability to survive within them. Ten bacterial strains tested--all of known oral infectivity--comprised Salmonella typhimurium, Listeria monocytogenes (three strains), Escherichia coli (two strains), Proteus mirabilis, Enterococcus faecalis, Bacteroides fragilis, and a Bacteroides sp. The phagocytic uptake of each strain was measured as the bacteria to phagocyte ratio after mononuclear phagocytes in mouse peritoneal exudate were permitted to ingest bacteria in vivo for 3 min. The three Listeria strains were the most susceptible to phagocytic uptake and the Salmonella strain was relatively resistant. The intracellular survival of each strain was studied during a subsequent 2 h in-vitro incubation of the mononuclear phagocytes that had been permitted to ingest bacteria in vivo. The strains with the best intracellular survival were Ent. faecalis and two of the three Listeria strains. The ability of S. typhimurium to survive intracellularly was intermediate but better than that of the two E. coli strains. Oral infectivity was not consistently correlated with susceptibility to ingestion by mononuclear phagocytes or ability to survive within them.

Benign pathologic responses of the larynx.

Benign laryngeal lesions were examined for patterns of injury indicated by deposition of fibronectin and collagen type IV. An immunoperoxidase technique was used to compare 33 fresh or paraffin-embedded tissues with regard to their staining of monoclonal antibodies directed against fibronectin and collagen type IV. Two types of patterns were recognized. One pattern showed intense fibronectin deposition in the superficial layer of the lamina propria, often coupled with basement membrane zone injury, indicated by thick collagen type IV bands. The other pattern showed rare basement membrane zone injury and very little fibronectin deposition. The first pattern correlated more with nodules, the second pattern more with Reinke's edema and some polyps. A better understanding of the effects of excessive deposition of structural glycoproteins such as fibronectin and of abnormal proteoglycan deposition may lead to a better characterization of vocal fold disease and its causation and, ultimately, better treatment.

Fever and the immune response. The effects of physiological temperatures on primary murine splenic T-cell responses in vitro.

Fever is a common sign of clinical disease but its biological purpose is unclear. Several cytokines mediate fever as well as the regulation of immune responses, and it was previously shown that murine thymocyte proliferative responses to IL-1 and IL-2 are exquisitely temperature-sensitive. The present studies were performed to determine whether such a high degree of temperature sensitivity was also a property of the primary in vitro immune responses of mature, murine splenic T cells. Using a physiological range of temperatures spanning those of normal skin (29 degrees C) to those of febrile core tissues (39 degrees C), primary murine spleen cell proliferative responses to either Con A, solid-phase anti-CD3 antibody, specific Ag, or allogenic cells were all found to display a high degree of temperature sensitivity. A similar pattern of strong temperature sensitivity was found for the capacity of primary MLR cultures to generate allospecific effector CTL. All of these forms of T-cell temperature sensitivity are expressed both in terms of the absolute degree of the response and in terms of its kinetics, the warmer temperatures up to 35 degrees C to 37 degrees C giving larger responses sooner than the lower physiological temperatures of 29 degrees C to 33 degrees C, at which these responses are markedly inhibited relative to those at 37 degrees C. In contrast, the effector cytolytic activity of a previously formed population of CTL is remarkably insensitive to the same temperature changes that strongly regulate the generation of those effector cells. The temperatures that usually define fever are those appropriate to the core tissues of a febrile animal, yet temperature changes in this range (37 degrees C-39 degrees C) had little stimulatory effect upon the T-cell responses studied, and were, in some cases, actually inhibitory. Temperature changes appropriate to the peripheral tissue temperatures in an animal becoming febrile (29 degrees C-37 degrees C) appear to be the ones that strongly regulate mature primary T-cell responses. These results suggest that one purpose of either fever or local tissue inflammation may be to temporarily ablate the cooler portions of normal thermal gradients, thereby selectively amplifying the emergence of T-cell immunity in peripheral tissues.

Temperature sensitivity of interleukin-dependent murine T cell proliferation: Q2 mapping of the responses of peanut agglutinin-negative thymocytes.

Recent studies in our laboratory have shown that IL 1-dependent mitogenic activity is present in apparently homogeneous preparations of rabbit endogenous pyrogen. This finding suggested that the mitogenic and pyrogenic activities of this molecule might serve a common goal. Initial studies on the temperature dependence of interleukin-dependent thymocyte mitogenesis suggested that high temperature sensitivity was associated with the action of IL 1 but not that of IL 2. The present study has used an expanded range of temperatures and refined tissue culture conditions to further examine the relative temperature dependence of thymocyte mitogenesis due to IL 1 or IL 2. Both the pI 5 and pI 7 species of rabbit IL 1 evoke highly temperature-sensitive responses from mouse thymocytes in the presence of a suboptimal dose of PHA and from peanut agglutinin-negative (PNA-) thymocytes in the absence of PHA. IL 2 also evokes a highly temperature-sensitive response from unseparated thymocytes in the presence of PHA. However, in the absence of PHA, vigorous responses by either unseparated or PNA- thymocytes to IL 2 alone lack strong temperature sensitivity. The temperature-dependent responses of both unseparated and PNA- thymocytes to either IL 1 or IL 2 have been analyzed by Q2 mapping, a determination of the temperature intervals most sensitive to temperature changes. By using this mode of analysis, we have found that IL 1 and IL 2 generate distinct Q2 maps, and that PHA transforms the shape of the IL 2-derived Q2 map but not that of IL 1. The possible significance of the temperature sensitivity of IL 1- and IL 2-driven reactions is discussed with respect to the biological functions of inflammation and fever.

Demonstration of interleukin 1 activity in apparently homogeneous specimens of the pI 5 form of rabbit endogenous pyrogen.

Rabbit mononuclear cells from oil-induced peritoneal exudates were purified by centrifugation on Percoll gradients, suspended in tissue culture medium, and stimulated with opsonized Staphylococcus epidermidis. The supernatants from these macrophages caused fever when injected intravenously into rabbits (endogenous pyrogen [EP] activity). The EP activity was contained in two protein fractions, with pIs of 7.3 and ca. 5.0. The same fractions caused mouse thymocytes to incorporate tritiated thymidine when incubated in vitro with small quantities of phytohemagglutinin (interleukin 1 [IL-1] activity). The pI 5.0 form of EP was purified to apparent homogeneity by sequential use of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, hydrophobic chromatography, and high-resolution isoelectric focusing. EP and IL-1 activities were not separable by any of these procedures. Active fractions from isoelectric focusing were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only one band was visible as judged by a silver staining method, and IL-1 activity could be recovered by renaturing eluates from the same region of sodium dodecyl sulfate gels run in parallel. An estimate of specific activity was made by comparing the intensity of stained bands of EP with the intensity of bands containing known quantities of lysozyme or RNase. By this criterion, the specific activity of purified pI 5 EP was between 17,000 and 58,000 degrees C U/mg of protein, and the specific activity in terms of IL-1 was between 59 million and 360 million U per mg of protein. These observations suggest that both EP and IL-1 activities can be expressed by a single molecular species. The implications of this coincidence are discussed. It was also shown that highly purified pI 5 EP obtained from macrophages stimulated in the presence of 14C-labeled amino acids contained significant 14C radioactivity. This suggests that the pI 5.0 EP, like the pI 7.3 form, was synthesized de novo from amino acid precursors.

Biochemical identification of citrobacteria in the clinical laboratory.

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).

The effects of variations in pH and temperature on the activation of mouse thymocytes by both forms of rabbit interleukin-1.

We studied the responses of unpurified mouse thymocytes and of peanut agglutinin-negative mouse thymocytes to the pI 7.3 form of rabbit Interleukin-1. We found that small increases of temperature strongly enhanced the mitogenic effect of this form of IL-1, and that the apparent temperature optimum was 37 degrees C. In both these respects the behavior of the pI 7.3 IL-1 resembled the previously described behavior of pI 5.0 IL-1. We suspected that the low apparent temperature optimum for IL-1 action was due to inadequate pH control by the bicarbonate-buffered medium. Experiments showed that small decreases in medium pH strongly inhibited the mitogenic action of both forms of IL-1. Furthermore, if thymocytes were stimulated with either form of IL-1 in strongly buffered media, the temperature optimum was at least 39 degrees C. The pI 7.3 and pI 5.0 forms of IL-1 are known to differ both biochemically and immunologically. Our experimental discovery that their temperature sensitivities are much the same suggests that temperature sensitivity is a property of the T cell rather than of the IL-1 molecules themselves.

The effect of temperature on the activation of thymocytes by interleukins I and II.

Endogenous pyrogen (EP) and interleukin (IL 1) appear to be closely related or identical. We therefore conducted experiments to test whether the central action of EP (fever) enhanced its local action (potentiation of immune responses). We found that 3H-TdR incorporation by thymocytes stimulated with IL 1 in the presence of 5 micrograms/ml of phytohemagglutinin (PHA) was strongly enhanced by small rises of temperature in the 32 to 38 degrees C range. Responses at 40 degrees C were generally less than those seen at 38 degrees C. Calculated values for the temperature coefficient (Q10) of the PHA-facilitated response to IL 1 varied between 50 and 300 in different experiments. Thymocytes had a variable response to IL 1 in the absence of PHA. However, thymocytes that failed to agglutinate with peanut agglutinin responded consistently to IL 1 alone, and these mitogenic responses were strongly temperature sensitive (Q10, 50 to 100 in different experiments). Thymocyte responses to 5 micrograms/ml PHA were only modestly sensitive to temperature (Q10, 3 to 8), and responses to interleukin 2 gave Q10 values of 3 to 24, with a mean of about 9. The responses of CTL cells to IL 2 were also modestly enhanced by small increases of temperature, and calculated Q10 values were similar to those observed for thymocytes. IL 1 production by macrophages was also not greatly enhanced by temperature (Q10, 2 to 5). These observations suggest that small increases of temperature enhance IL 1 activity to a much greater degree than they enhance the activity of other components of the immune response. This may help to explain the conservation of two such apparently disparate activities as EP and IL 1 in the same molecules.

Escherichia coli O157:H7 generates a unique biochemical profile on MicroScan conventional gram-negative identification panels.

More than 90% of the strains of Escherichia coli O157:H7 that were identified on a MicroScan gram-negative dried conventional (overnight) panel gave one of two unique biochemical profile numbers that were not detected in other d-sorbitol-negative E. coli or in other strains isolated from pathogenic processes. This suggests that the panel has the capability of being used as a preliminary screening tool for O157:H7 strains involved in hemorrhagic colitis when MacConkey-sorbitol agar is not available.