Fc gamma R expression on NK cells influences disease severity in rheumatoid arthritis

Rheumatoid arthritis (RA) is associated with autoantibodies, the best known of which is rheumatoid factor (RF). RF/IgG complexes interact with FcgammaR on the surface of neutrophils, NK cells and monocyte/macrophages. We have analyzed the expression pattern and allelic polymorphisms of three FcgammaR genes (FcgammaRIIA, FcgammaRIIC and FcgammaRIIIA) in a large sample of RA patients and normal donors. We have found that the level of FcgammaR (CD16 and CD32) expression on NK cells is lower in RA patients than in normal individuals. Genotypic analysis demonstrated that the CD32 isoform expressed by the majority of RA patients was not the activating FcgammaRIIc1 isoform, commonly seen in normal individuals, but rather the inhibitory FcgammaRIIb isoform. The combination of the FcgammaRIIIA-176F allele with a lack of CD32 expression in NK cells appeared to be characteristic of RA subjects with aggressive disease. Since FcgammaRII and FcgammaRIIIA are predominantly expressed by NK cells, these data further suggest that FcgammaR-mediated activation of NK cells could be a disease-determining factor in RA patients.     

Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation

The effects of granulocyte-macrophage colony-stimulating factor (GM- CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T- cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM- CSF responsive cell type, thymic AC and T cells were examined for GM- CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single- cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.     

Effects of exogenous estrogen on uterine leukocyte recruitment

OBJECTIVE: To determine the role of estrogen in leukocyte recruitment to the human endometrium. DESIGN: Prospective, controlled in vivo study. SETTING: Academic research laboratory. PATIENT(S): Ten patients presenting for donor oocytes. INTERVENTION(S): Endometrial biopsies for the evaluation of leukocyte populations were collected from perimenopausal women in two consecutive regulated cycles who were given two different regimens of estrogen with identical progesterone treatment. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of endometrial leukocyte populations and relative levels of expression of three chemokine genes. RESULT(S): The total uterine leukocyte population increased significantly when the women received oral estrogen, which resulted in higher serum estrogen levels. This rise in leukocytes was due to a significant increase in both the uterine natural killer cells and the macrophage populations. T-cell numbers did not change relative to circulating estrogen levels. The relative abundance of mRNA from three chemokines was also determined. No changes were found in the expression of M-CSF or MCP-1. Interleukin 8 decreased in glands relative to estrogen levels. CONCLUSION(S): These data demonstrate that changes in circulating levels of estrogen can regulate the recruitment of bone marrow-derived cells to the uterine endometrium; however, the mechanism whereby that occurs remains elusive.     

Macrophage phenotype as a determinant of biologic scaffold remodeling

Macrophage phenotype can be characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). The present study used a rat model to determine the macrophage phenotype at the site of implantation of two biologic scaffolds that were derived from porcine small intestinal submucosa (SIS) and that differed mainly according to their method of processing: the Restore device (SIS) and the CuffPatch device (carbodiimide crosslinked form of porcine-derived SIS (CDI-SIS)). An autologous tissue graft was used as a control implant. Immunohistologic methods were used to identify macrophage surface markers CD68 (pan macrophages), CD80 and CCR7 (M1 profile), and CD163 (M2 profile) during the remodeling process. All graft sites were characterized by the dense population of CD68+ mononuclear cells present during the first 4 weeks. The SIS device elicited a predominantly CD163+ response (M2 profile, p < 0.001) and showed constructive remodeling at 16 weeks. The CDI-SIS device showed a predominately CD80+ and CCR7+ response (M1 profile, p < 0.03), and at 16 weeks was characterized by chronic inflammation. The autologous tissue graft showed a predominately CD163+ response (M2) at 1 week, with a dual M1/M2 population (CD80+, CCR7+, and CD163+) by 2 and 4 weeks and moderately well organized connective tissue by 16 weeks. The processing methods used during the manufacturing of a biologic scaffold can have a profound influence upon the macrophage phenotype profile and downstream remodeling events. Routine histologic examination alone is inadequate to determine the phenotype of mononuclear cells that participate in the host response to the scaffold.     

Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.     

Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti- IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)- induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.     

Degradation and remodeling of small intestinal submucosa in canine Achilles tendon repair

BACKGROUND: Extracellular matrix derived from porcine small intestinal submucosa is used for the repair of musculotendinous tissues. Preclinical evaluation and clinical use have suggested that small intestinal submucosa extracellular matrix degrades rapidly after implantation and can be replaced by host tissue that is functionally and histologically similar to the normal tissue. METHODS: The present study analyzed the temporal degradation of a ten-layer multilaminate device of small intestinal submucosa extracellular matrix used for the repair of canine Achilles tendon and examined the corresponding histological appearance of the remodeled tissue during the course of scaffold degradation. Devices were fabricated from small intestinal submucosa extracellular matrix labeled with 14C. The amount of 14C remaining in the remodeled graft was measured by liquid scintillation counting at three, seven, fourteen, twenty-eight, sixty, and ninety days after surgery. Blood, urine, feces, and other parenchymal tissues were also harvested to determine the fate of scaffold degradation products. Tissue specimens were prepared for routine histological analysis to examine the morphology of the remodeled graft at each time-point. RESULTS: The small intestinal submucosa extracellular matrix graft degraded rapidly, with approximately 60% of the mass lost by one month after surgery, and the graft was completely resorbed by three months after surgery. The graft supported rapid cellular infiltration and host tissue ingrowth. By ninety days after surgery, the remodeled small intestinal submucosa extracellular matrix consisted of a dense collagenous tissue with organization, cellularity, and vascularity similar to that of normal tendon. CONCLUSIONS: Small intestinal submucosa extracellular matrix is rapidly degraded after implantation for the repair of a musculotendinous tissue in this canine Achilles tendon repair model and is replaced by the deposition and organization of host tissue that is histologically similar to that of normal tissue.