Differences in innate immunologic response to group B streptococcus between colonized and noncolonized women.

OBJECTIVE: To evaluate the functional capacity of granulocytes and monocytes from pregnant and nonpregnant women in relation to group B streptococcus (GBS) colonization status. METHODS: Engulfment of fluorescent GBS by peripheral blood phagocytes from GBS-colonized and noncolonized women was measured by flow cytometry. Intracellular superoxiode generated in response to GBS challenge to monocytes and granulocytes enriched from peripheral blood of these women was also measured by flow cytometry, and extracellular superoxide was determined by colorimetric assay. RESULTS: Monocytes and granulocytes from pregnant, GBS-colonized women engulfed significantly greater numbers of GBS than phagocytes from pregnant, noncolonized women. No difference in intracellular superoxide production was detected between any of the groups of women; however, monocytes from pregnant, colonized women released significantly more superoxide into the extracellular milieu than did granulocytes from the same women. No differences in extracellular release of superoxide were observed among noncolonized women whether they were pregnant or not. CONCLUSIONS: Monocytes from pregnant, colonized women engulf more GBS and release more of the superoxide into the extracellular environment, where it is unlikely to be an effective defense mechanism against intracellular bacteria. This suggests that components of the innate immune system that should serve in a protective role may function suboptimally, thereby contributing to the colonization process by GBS.

Differential expression of chemokines in a mouse model of wound healing.

Macrophages have a multifaceted role in wound healing. While their initial activity may be in the degradation and elimination of damaged tissue, macrophages also produce and secrete a variety of mediators that can participate in the repair process as well. To perform these functions, macrophages must be recruited to a wound site. Our purpose was to examine the temporal and spatial expression of macrophage chemoattracting cytokines (chemokines) at a surgical wound site. A surgical wound was prepared on the dorsal aspect of B6AF1/J mice. Biopsies were obtained from the wound and a comparable nonwounded area between 6 and 72 hr after wounding. The presence or absence of various chemokine mRNAs was detected by the reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemical staining and in situ RT-PCR determined localization of cells producing chemokines. In wounded tissue, both macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1) were detected; however, the time of expression differed for each molecule. MCP-1 mRNA was detected at 6 hr after wounding, with decreased expression at subsequent time periods. In contrast, MIP-1 messages were not observed until 24 hr after wounding, and steadily increased thereafter. MCP-1 and MIP-1 mRNA and protein were localized predominantly in keratinocytes. The rapid and strong expression of MCP-1 and MIP-1 messages within the wound site suggests a pivotal role for these chemokines in the repair process. The differences in appearance and level of expression over time, however, suggest distinctive functions for each chemokine and indicate that the local milieu, rather than a single cytokine, influences macrophage recruitment and/or activation.

Adoptive transfer of skin-selective autoimmunity induced by Skn alloantigenic disparities.

Two unlinked genes of the mouse, Skn-1 and Skn-2, each with alterative alleles, specify alternative cell-surface Skn alloantigens expressed only by epidermal and neural cells. C57BL/6 (B6) and A/J (A) strain mice differ at both Skn loci. Thus lethally irradiated B6 mice restored with (B6 x A)F1 hybrid hematopoietic cells [(B6 x A)/B6 chimeras] reject A strain (Skn-incompatible) skin grafts. Our studies were designed primarily to test the inference that (B6 x A)F1 lymphoid cells, after differentiating in B6 recipients, which lack the Skn alloantigens of A strain mice, may make an Skn-related, skin-selective autoimmune response when returned to their native (B6 x A)F1 habitat. Severe cutaneous lesions did, indeed, ensue after spleen cells of (B6 x A)/B6 chimeras were transferred to (B6 x A)F1 recipients, provided that three conditions were met--namely, (i) priming of the (B6 x A)/B6 chimeric donor by grafting and rejection of Skn-incompatible A strain skin grafts, (ii) stimulation of the recipient's skin as from shaving, at which sites the lesions were mainly located, and (iii) pretreatment of the (B6 x A)F1 recipients with cyclophosphamide or sublethal irradiation. Spleen cells of control female chimeras primed by grafting and rejection of H-Y (Skn-compatible) B6 male skin failed to incite the Skn-typical cutaneous lesions in (B6 x A)F1 recipients, indicating that these lesions were Skn-specific and not a nonspecific consequence of incompatible skin grafting per se. Normally compatible A strain skin grafts, but not Skn-compatible B6 skin grafts, were rejected by cyclophosphamide-treated (B6 x A)F1 recipients of (B6 x A)/B6 spleen cells from Skn-primed chimera donors. Treatment of primed chimeras' spleen cells with antiserum to H-2a (A strain) specifically abolished their capacity to adoptively incite the Skn-related autoimmune syndrome, confirming that the immune cells responsible are of (B6 x A)F1 origin and are not residual B6 derivatives. These findings add weight to the status of Skn systems as agents of tissue-selective histoincompatibility and, perhaps, of clinical disorders with a known or suspected autoimmune basis affecting the skin.

Immunohistochemical identification of Skn antigens in mouse epidermis.

Rejection of murine skin grafts by hematopoietic chimeras that are fully compatible genetically with the skin-graft donor has been attributed to a disparity in skin-selective alloantigens between the irradiated host and the skin graft donor. Monoclonal antibodies recognizing Skna alloantigen were produced and used in indirect immunofluorescent and immunoperoxidase tests with sections of skin to demonstrate and confirm the expression of Skna alloantigen on epidermal cells of Skna genotype and absence of Skna alloantigen from epidermal cells of non-Skna genotype.

Demonstration of Skn antigens on mouse epidermal cells by immunofluorescence and flow cytometry.

C57BL/6 (B6) mice that were lethally irradiated and reconstituted with (B6XA/J)F1 spleen cells were immunized against the skin-specific antigens, Skn, by grafting with A/J tail skin. Serum from these mice was shown to contain Skna-specific antibody by a flow cytometric assay using indirect immunofluorescence.

In vitro adherence of Moraxella bovis to intact corneal epithelium.

An in vitro assay is described using radiolabeled Moraxella bovis for studying adherence to intact bovine corneal epithelial surfaces. The assay was optimized for time (45 min) and for the ratio of epithelial cells to bacteria (1:1000) that demonstrated a significant difference in adherence between M. bovis strain 118F, a piliated organism and a nonpiliated variant, strain 118F/4-2. Adherence of these organisms correlated with previous pathogenicity studies involving experimental infection of calves. Scanning electron microscopy of tissues treated in the assay revealed a predilection of M. bovis for dark epithelial cells and for association with depressions in the tissue surface. This assay technique is discussed in comparison with other in vitro adherence assay methods.