There is considerable evidence that suppression of the immune system by UVB (280-320 nm UV) irradiation is initiated by UVB-dependent isomerization of a specific skin photoreceptor, urocanic acid (UCA), from the trans to the cis form. Previous studies have confirmed that cis-UCA administration to mice 3-5 days prior to hapten sensitization at a distant site, suppresses the contact hypersensitivity (CHS) response upon challenge. This study demonstrates in mice that cis-UCA, like UVB, suppresses CHS to trinitrochlorobenzene by a mechanism partly dependent on prostanoid production. In vitro experimentation showed that human keratinocytes, isolated from neonatal foreskin, increased prostaglandin E2 (PGE2) production in response to histamine but not UCA alone. However, cis-UCA synergized with histamine for increased PGE2 production by keratinocytes. Cis-urocanic acid also increased the sensitivity of keratinocytes for PGE2 production in response to histamine. Prostaglandin E2 from keratinocytes exposed to cis-UCA and histamine may contribute directly, or indirectly, to the regulation of CHS responses by UVB irradiation.

Intraabdominal abscesses were induced in mice by intraperitoneal inoculation of Bacteroides fragilis and Escherichia coli plus bran as the abscess-potentiating agent. Sixor seven-day-old abscesses were mechanically disaggregated in buffer, and the cells obtained were fractionated on discontinuous Percoll density gradients. Neutrophil populations of different density, each approximately 90% pure, were isolated. When the abscess-derived neutrophils were subsequently incubated with normal serum in vitro under aerobic conditions, the viability of the gram-negative bacteria that had been phagocytosed within the abscess did not change significantly. This anergy to intracellular bacteria (on subsequent incubation in vitro under optimal conditions for phagocytic killing) was also found for neutrophils that had been obtained from abscesses induced by a mixture that included Proteus mirabilis plus B. fragilis and from those induced by E. coli plus P. mirabilis. While unable to significantly kill intracellular organisms that had been phagocytosed in vivo, the abscessderived neutrophils could engulf and kill organisms to which they were exposed in vitro. Neutrophils from abscesses induced by P. mirabilis only plus bran killed that organism introduced in vitro significantly more effectively than the organisms that had been engulfed in vivo. In contrast, neutrophils from abscesses induced by the gram-positive organism Staphylococcus aureus plus bran were able to kill their intracellular organisms on subsequent incubation in vitro as effectively as they could kill added S. aureus. Neutrophils isolated from the peripheral blood and from induced peritoneal exudates of abscess-bearing mice were able to phagocytose and kill organisms in vitro with greater efficiency than abscess-derived neutrophils. The mechanism whereby neutrophils from abscesses induced by the gram-positive organism S. aureus can kill the organisms phagocytosed in vivo on subsequent in vitro incubation, in contrast to the relative anergy to their intracellular organisms displayed by neutrophils derived from abscesses induced by combinations of gram-negative bacteria, is not known.

Bacteroides fragilis and Escherichia coli are synergistic in the production of intraabdominal abscesses. However, these bacteria initiate abscess formation only when inoculated with an agent such as autoclaved colonic contents (ACC) or bran (a fiber analogue). The mechanism of action of the abscess-potentiating agent was studied. Opsonins in normal mouse serum were determined for phagocytic killing by murine neutrophils of B. fragilis and E. coli. Opsonization required fixation of complement by the alternative pathway. ACC (0.2 mg/ml) and bran (1.0 mg/ml) inhibited phagocytic killing of Proteus mirabilis in the presence of normal but not immune serum. Assay of the alternative pathway of complement activation indicated that both bacterial components and abscess-potentiating agents in an abscess-inducing mixture activated complement. These findings suggest that abscess-potentiating agents inhibit opsonization and therefore the subsequent phagocytic killing of bacteria in the nonimmune host.

The role of the Fc and third component of complement (C3) receptors on mouse neutrophils in the control of killing of Proteus mirabilis, opsonized in normal mouse serum (NMS) or heated immune mouse serum (HIMS), was studied. The events following incubation of neutrophils with P. mirabilis and the events associated with bacterial killing were assayed. The respiratory burst was quantified by chemiluminescence (CL). Levels of leukocyte-associated bacteria were determined after a 20-min ingestion period as a measure of phagocytosis. Bacterial killing was measured while ingestion was allowed to continue or as a discrete process when extracellular, noningested bacteria had been removed and neutrophils with intracellular bacteria were incubated in the presence of serum. Modification of these responses in the presence of three monoclonal antibodies (MAb), NIMP-R10 and M1/70, which bind to different epitopes of the mouse C3 receptor, and 2.4G2, which binds to the mouse Fc receptor, was investigated. MAb to the C3, but not to the Fc, receptors reduced CL, ingestion, and intracellular killing of NMS-opsonized P. mirabilis. MAb to the Fc receptor diminished CL to and reduced the rate of ingestion of HIMS-opsonized bacteria. The two MAb to the C3 receptor each produced a similar inhibition of ingestion and intracellular killing of HIMS-opsonized bacteria, but they only partially blocked CL. A range of MAb preparations reactive with other murine antigens did not inhibit these events, either with NMS- or HIMS-opsonized P. mirabilis. The results suggest that C3 receptors on mouse neutrophils played a predominant role in regulation of the killing of P. mirabilis. Similar results were found for Staphylococcus aureus. C3 receptors were necessary for maximal expression of all functions culminating in bacterial kill. That MAb to the C3 receptor inhibited phagocytosis of HIMS-opsonized bacteria in similar fashion to the effect of MAb to the Fc receptor and in contrast to the lack of effect of control MAb may reflect steric hindrance of the Fc receptor by MAb binding to the C3 receptor, or it may reflect that the receptors are linked in murine neutrophils as they are in human neutrophils.

Summary. In the absence of antimicrobial therapy, bacteria such as Bacteroides fragilis, Escherichia coli and Proteus mirabilis may persist within an intra-abdominal abscess in the presence of large numbers of neutrophils which, under optimal conditions in vitro, can readily phagocytose and kill the same bacterial strains. Neutrophils taken from abscesses induced by gram-negative bacteria such as those above contain viable organisms. On incubation in vitro in the presence of serum, these neutrophils kill the bacteria phagocytosed in the abscess poorly, if at all, yet can readily kill organisms added in vitro. To determine possible mechanisms that might explain this, we examined the bactericidal activity in vitro of neutrophils from a range of abscesses induced by one or two species of bacteria plus an abscess-potentiating agent, bran. The organisms studied were B. fragilis, E. coli, P . mirabilis and Staphylococcus aureus. The killing in vitro of E. coli and P . mirabilis, engulfed within an abscess, was significantly less than that of the same organisms when they were added to the in-vitro assay. In contrast, the killing of S. aureus was similar, whether engulfed in vivo or in vitro. However, S. aureus was less susceptible to phagocytosis and killing in vitro than P . mirabilis or E. coli, and the killing of S. aureus during in-vitro incubation of neutrophils that had engulfed the organism within the abscess was similar to that of the gram-negative bacteria engulfed within the abscess. We examined whether organisms phagocytosed in vivo were within neutrophils which had diminished or no expression of cell surface Fc and C3 receptors, and might, therefore, be unresponsive to extracellular serum factors such as complement which stimulate intracellular killing. No population of neutrophils lacking CR3 complement receptors was detected. Furthermore, the expression of these receptors was enhanced on abscess neutrophils compared with peripheral blood neutrophils. FcyRII receptor expression was equivalent in the two populations. Extracellular serum was necessary for any abscess neutrophil bactericidal activity. To determine whether the bacteria within abscess neutrophils were a subpopulation selected for resistance to intracellular killing, we lysed the neutrophils and added the released bacteria to bactericidal assays with peritoneal exudate neutrophils. The bacteria were killed readily, but not as efficiently as log-phase organisms grown in vitro. As these studies have shown that neutrophils from abscesses are functional, with enhanced CR3 expression, and that bacteria persisting within abscesses are not resistant to intracellular killing, it is possible that the poor killing in vitro of bacteria engulfed in uivo by abscess neutrophils reflects depleted intracellular killing mechanisms in those particular neutrophils, or results from the phagocytosis of organisms under conditions prevailing within abscesses, or is due to as yet undefined bacterial virulence factors.